U.S. patent number 7,811,602 [Application Number 11/131,436] was granted by the patent office on 2010-10-12 for liposomal formulations comprising dihydrosphingomyelin and methods of use thereof.
This patent grant is currently assigned to Tekmira Pharmaceuticals Corporation. Invention is credited to Steven M Ansell, Pieter R Cullis, Michael J Hope, Thomas D Madden, Norbert Maurer, Barbara L S Mui, Sean C Semple.
United States Patent |
7,811,602 |
Cullis , et al. |
October 12, 2010 |
**Please see images for:
( Certificate of Correction ) ** |
Liposomal formulations comprising dihydrosphingomyelin and methods
of use thereof
Abstract
The present invention includes novel liposomes comprising
dihydrosphingomyelin. The invention also includes compositions
comprising these liposomes and a therapeutic agent, in addition to
methods and kits for delivering a therapeutic agent or treating a
disease, e.g., a cancer, using these compositions.
Inventors: |
Cullis; Pieter R (Vancouver,
CA), Madden; Thomas D (Vancouver, CA),
Hope; Michael J (Vancouver, CA), Ansell; Steven M
(Vancouver, CA), Mui; Barbara L S (Vancouver,
CA), Semple; Sean C (Vancouver, CA),
Maurer; Norbert (Vancouver, CA) |
Assignee: |
Tekmira Pharmaceuticals
Corporation (Burnaby, CA)
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Family
ID: |
35169836 |
Appl.
No.: |
11/131,436 |
Filed: |
May 16, 2005 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20060008909 A1 |
Jan 12, 2006 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60631997 |
Nov 30, 2004 |
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60571712 |
May 17, 2004 |
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Current U.S.
Class: |
424/450 |
Current CPC
Class: |
A61K
9/1272 (20130101); A61K 31/4745 (20130101); A61K
33/32 (20130101); A61K 31/4745 (20130101); A61K
2300/00 (20130101); A61K 33/32 (20130101); A61K
2300/00 (20130101); A61K 9/1278 (20130101) |
Current International
Class: |
A61K
9/127 (20060101) |
Field of
Search: |
;424/450 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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WO 91/17424 |
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Nov 1991 |
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WO |
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WO 95/08986 |
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Apr 1995 |
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WO |
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WO 02/072010 |
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Sep 2002 |
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WO |
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Primary Examiner: Kishore; Gollamudi S
Attorney, Agent or Firm: Seed IP Law Group PLLC
Parent Case Text
CROSS-REFERENCES TO RELATED APPLICATIONS
This application claims the benefit under 35 U.S.C. .sctn.119(e) of
U.S. Provisional Patent Application No. 60/571,712, filed May 17,
2004; and U.S. Provisional Patent Application No. 60/631,997, filed
Nov. 30, 2004, where these provisional applications are
incorporated herein by reference in their entireties.
Claims
The invention claimed is:
1. A liposomal composition comprising a liposome, wherein said
liposome comprises phospholipid and cholesterol at a molar ratio
from 75/25 (mol/mol) total phospholipid/cholesterol to 25/75
(mol/mol) total phospholipid/cholesterol, wherein said at least 20%
(molar basis) of total phospholipid present in said liposome is
dihydrosphingomyelin (DHSM), wherein the interior of said liposome
comprises MnSO.sub.4 or MgSO.sub.4, and wherein said liposome
comprises a therapeutic agent.
2. The liposomal composition of claim 1, wherein said DHSM
constitutes at least 50% (molar basis) of total phospholipid
present in said liposome.
3. The liposomal composition of claim 1, wherein the N-acyl chain
of said DHSM consists of 12 to 24 carbon atoms.
4. The liposomal composition of claim 1, wherein the DHSM is
selected from the group consisting of:
N-palmitylsphinganyl-1-O-phosphorylcholine;
N-stearylsphinganyl-1-O-phosphorylcholine;
N-myristylsphinganyl-1-O-phosphorylcholine; and
N-arachidylsphinganyl-1-O-phosphorylcholine.
5. The liposomal composition of claim 1, wherein the DHSM N-acyl
and dihydrosphingosine chains comprise carbon chains that are not
different in length by more than four carbon atoms.
6. The liposomal composition of claim 1, wherein the therapeutic
agent is an antineoplastic agent.
7. The liposomal composition of claim 6, wherein the antineoplastic
agent is selected from the group consisting of: vinca alkaloids,
camptothecins, etoposide, and taxanes.
8. The liposome composition of claim 1, further comprising empty
liposomes.
9. A method of delivering a therapeutic agent to a patient,
comprising administering to the patient a pharmaceutical
composition comprising a liposome-encapsulated therapeutic agent,
wherein said liposome comprises phospholipid and cholesterol at a
molar ratio from 75/25 (mol/mol) total phospholipid/cholesterol to
25/75 (mol/mol) total phospholipid/cholesterol, wherein at least
50% (molar basis) of total phospholipid present in said liposome is
dihydrosphingomyelin (DHSM), and wherein the interior of said
liposome comprises MnSO.sub.4 or MgSO.sub.4.
10. The method of claim 9, wherein the therapeutic agent is an
antineoplastic agent.
11. The method of claim 10, wherein the antineoplastic agent is
selected from the group consisting of: vinca alkaloids,
camptothecins, etoposide, and taxanes.
12. A method of treating a cancer in a mammal, comprising
administering to the mammal a pharmaceutical composition comprising
a liposome-encapsulated therapeutic agent, wherein said liposome
comprises phospholipid and cholesterol at a molar ratio from 75/25
(mol/mol) total phospholipid:cholesterol to 25/75 (mol/mol) total
phospholipid:cholesterol, wherein at least 50% of total
phospholipids present in said liposome is dihydrosphingomyelin
(DHSM), and wherein the interior of said liposome comprises
MnSO.sub.4 or MgSO.sub.4.
13. The method of claim 12, wherein the cancer is a leukemia or
lymphoma.
14. The method of claim 12, wherein the cancer is a solid
tumor.
15. A method of producing a pharmaceutical composition, comprising
loading a liposome comprising phospholipid id and cholesterol at a
molar ratio from 75/25 (mol/mol) total phospholipid:cholesterol to
25/75 mol/mol total phospholipid:cholesterol with a therapeutic
agent, wherein at least 50% of the total phospholipids present in
said liposome is dihydrosphingomyelin (DHSM), and wherein the
interior of said liposome comprises MnSO.sub.4 or MgSO.sub.4.
16. The method of claim 15, wherein said liposome comprises a
buffer containing MnSO.sub.4 at a concentration equal to or greater
than 300 mM, wherein said loading is performed at a temperature
equal to or greater than 60.degree. C. and in the presence of an
ionophore.
17. A method of loading a therapeutic agent into a liposome
comprising: incubating a liposome comprising phospholipid and
cholesterol at a molar ratio from 75/25 (mol/mol) total
phospholipid:cholesterol to 25/75 (mol/mol) total
phospholipid:cholesterol and having an encapsulated medium
comprising MnSO.sub.4 or MgSO.sub.4, wherein at least 50% of the
total phospholipid of the liposome is dihydrosphingomyelin (DHSM),
with an external solution comprising said therapeutic agent and an
ionophore at a temperature greater than 60.degree. C. to form a
therapeutic agent-loaded liposome.
18. A kit comprising: (a) a liposome comprising phospholipid and
cholesterol at a molar ratio from 75/25 (mol/mol) total
phospholipid:cholesterol to 25/75 (mol/mol) total
phospholipid:cholesterol, wherein said at least 50% of total
phospholipid present in said liposome is dihydrosphingomyelin
(DHSM), and wherein the interior of said liposome comprises
MnSO.sub.4 or MgSO.sub.4, and (b) a therapeutic agent.
Description
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to liposomes, liposomal compositions,
and methods suitable for the delivery of active agents.
2. Description of the Related Art
A major challenge facing medical science and the pharmaceutical
industry, in particular, is to develop methods for providing
therapeutic agents to appropriate tissues or cells at a sufficient
dosage to provide a therapeutic benefit, without prohibitively
harming the patient being treated. Accordingly, it is an important
goal of the pharmaceutical industry to develop drug delivery
devices and methods that provide increased efficacy with decreased
associated toxicity. A variety of different general approaches have
been taken, with various degrees of success. These include, e.g.,
the use of implantable drug delivery devices, the attachment of
targeting moieties to therapeutic compounds, and the encapsulation
of therapeutic compounds in carriers, e.g., liposomes, to modulate
drug biodistribution and the duration of drug exposure.
Liposomes are particulate carriers and, hence, tend to remain
within the blood compartment, as they are not able to extravasate
across the continuous endothelial lining present in most blood
vessels. At disease sites, however, the blood vessels may be leaky,
allowing liposome extravasation and accumulation in the
interstitial space. In tumors, for example, the immature
neovasculature tends to exhibit pores or defects that can allow
liposomes of appropriate size to exit the blood vessels (Yuan et
al., Cancer Research 54: 3352-3356, 1994). Similarly, at sites of
infection or inflammation, the endothelial permeability barrier can
be compromised, allowing liposomes to accumulate in these regions.
In contrast, the blood vessels present in most normal, healthy
tissues tend to have continuous endothelial linings. Hence,
liposomal delivery can reduce drug exposure to these areas.
Exceptions are the organs of the mononuclear phagocyte system
(MPS), such as the liver and spleen, where fenestrated capillaries
are present.
In efforts to develop more effective therapeutic treatments, a
variety of compounds have been formulated in liposomes. For
example, many anticancer or antineoplastic drugs have been
encapsulated in liposomes. These include vinca alkaloids,
alkylating agents, nitrosoureas, platinum co-ordination complexes,
antimetabolites, anthracyclines, and camptothecins. Studies with
liposomes containing anthracycline antineoplastics have clearly
shown reduction of cardiotoxicity and prolonged survival of tumor
bearing animals compared to controls receiving free drug. In
addition, liposomal formulations of antibiotics, anti-inflammatory
agents, and antifungal drugs have been described.
In order to achieve efficient drug delivery to disease sites using
liposomal carriers, however, the liposomes should exhibit a
relatively long plasma circulation half-life to increase the
likelihood of extravasate during passage through the site. In
addition, drug release from the liposomes should be slow to reduce
drug loss prior to carrier accumulation at the disease site.
Further, drug activity is often dependent on the duration of drug
exposure. In order to optimize efficacy, therefore, slow drug
release from the liposomes may be required.
Considerable efforts have been made to identify liposomal carrier
compositions that show slow clearance from the blood, and
long-circulating carriers have been described in numerous
scientific publications and patents. Such long-circulating carriers
may employ polymer coatings, e.g., polyethylene glycol (PEG), to
reduce uptake by the MPS (reviewed by Allen and Stuart in
Liposomes: Rationale Design, Janoff, A. S. (ed), Marcel Dekker
Inc., New York (1999); Allen et al., Biochimica et Biophysica Acta
1066: 29-36, 1991) or may employ specific lipid compositions, such
as ganglioside (U.S. Pat. No. 4,837,028; Allen and Choon, FEBS
Letters, 223: 4246, 1987), or sphingomyelin and cholesterol (U.S.
Pat. No. 5,543,152; U.S. Pat. No. 5,741,516; U.S. Pat. No.
5,814,335). Efforts have also been made to control drug leakage or
release rates from liposomal carriers, using for example, various
lipid components or a transmembrane potential to control release
(U.S. Pat. No. 5,077,056). Alternatively, drug release rates may be
controlled by precipitation of the drug within the liposomal
carriers (U.S. Patent Publication No. 2002/0119990-A1) as indicated
by the previous studies, not all lipid formulations are equal for
drug delivery purposes and extensive research continues into
formulations which demonstrate preferred characteristics for drug
loading and storage, drug administration, pharmacokinetics,
biodistribution, leakage rates, tumor accumulation, toxicity
profile, and the like. Accordingly, while numerous liposomes and
lipid-based drug delivery vehicles have been developed, there is
clearly still a need in the art for improved liposomal
compositions, including liposomes that provide reduced levels of
clearance and slow drug release.
BRIEF SUMMARY OF THE INVENTION
The present invention includes liposomes, liposomal compositions
and related methods and kits. In one embodiment, the invention
provides a liposome comprising dihydrosphingomyelin (DHSM) wherein
said DHSM constitutes at least 20% or at least 50% of total
phospholipid present in said liposome.
In various embodiments of liposomes of the invention, the DHSM
N-acyl chain consists of 12 to 24 carbon atoms. In one particular
embodiment, the DHSM N-acyl chain consists of 16 carbon atoms.
In other related embodiments, the DHSM is selected from the group
consisting of: D-erythro-N-palmityl-dihydrosphingomyelin
(16:0-DHSM), D-erythro-N-stearyl-dihydrosphingomyelin (18:0-DHSM),
D-erythro-N-arachidyl-dihydrosphingomyelin,
D-erythro-N-heneicosanyl-dihydrosphingomyelin,
D-erythro-N-behenyl-dihydrosphingomyelin,
D-erythro-N-tricosanyl-dihydrosphingomyelin,
D-erythro-N-lignoceryl-dihydrosphingomyelin. Alternatively the DHSM
may consists of a mixture of N-acyl chains, such as the mixture of
N-acyl chains present in brain sphingomyelin, egg sphingomyelin or
milk sphingomyelin, or such mixtures of N-acyl chains derived from
such natural sources but where any unsaturated N-acyl chain is
saturated. In particular embodiments, the DHSM is brain DHSM, egg
DHSM, or milk DHSM.
In one embodiment, the DHSM is prepared by hydrogenation of a
synthetic sphingomyelin. In another embodiment the DHSM is prepared
by hydrogenation of a natural sphingomyelin, such as brain
sphingomyelin, egg sphingomyelin or milk sphingomyelin.
In yet another embodiment, the DHSM N-acyl and sphingosine chains
comprise carbon chains that are not different in length by more
than four carbon atoms.
In a further related embodiment, at least 50% of the DHSM comprises
DHSM wherein the N-acyl and dihydrosphingosine comprise carbon
chains that are not different in length by more than four carbon
atoms. In certain embodiments of the invention, the liposomes
further comprise cholesterol. In particular embodiments, the DHSM
and cholesterol are present at a molar ratio from 75/25 (mol/mol)
DHSM/cholesterol to 25/75 (mol/mol) DHSM/cholesterol. In particular
embodiments, the DHSM and cholesterol are present at a molar ratio
from 60/40 (mol/mol) DHSM/cholesterol to 40/60 (mol/mol)
DHSM/cholesterol. In further embodiments, the DHSM and cholesterol
are present at a molar ratio of either about 55/45 (mol/mol) or
about 50/50 (mol/mol) DHSM/cholesterol.
In certain embodiments of the invention, the liposomes comprise
DHSM, cholesterol and other phospholipids or derivatized
phospholipids, wherein DHSM comprises at least 20% or at least 50%
of the total phospholipid present, and cholesterol is present at a
molar ratio from 75/25 (mol/mol) total phospholipid/cholesterol to
25/75 (mol/mol) total phospholipid/cholesterol. In particular
embodiments, the liposomes comprise DHSM, cholesterol and other
phospholipids or derivatized phospholipids, wherein DHSM comprises
at least 20% or at least 50% of the total phospholipid present, and
cholesterol is present at a molar ratio from 60/40 (mol/mol) total
phospholipid/cholesterol to 40/60 (mol/mol) total
phospholipid/cholesterol. In particular embodiments, total
phospholipid and cholesterol are present at a molar ratio of about
55/45 (mol/mol) total phospholipid/cholesterol or about 50/50
(mol/mol) total phospholipid/cholesterol.
In another embodiment, more than 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80% or 90% of the DHSM N-acyl chains are saturated.
In other embodiments, the invention provides a liposomal
composition comprising a liposome of the invention and a
therapeutic compound.
In one embodiment, the therapeutic compound is an antineoplastic
agent. In specific embodiments, the antineoplastic agent is a vinca
alkaloid, a camptothecin, an anthracycline, NK611, an etoposide, or
a taxane. In particular embodiments, the vinca alkaloid is
vincristine, vinblastine, or vinorelbine. In other embodiments, the
camptothecin is topotecan or irinotecan, or SN-38. In other
embodiments, the taxane is paclitaxel or docetaxel.
In yet another related embodiment, the invention includes methods
of delivering a therapeutic agent to a patient, comprising
administering to the patient a pharmaceutical composition
comprising a liposome-encapsulated therapeutic agent, wherein said
liposome comprises DHSM and wherein at least 20% or at least 50% of
the total phospholipids present in said liposome is DHSM. In one
embodiment, the liposome used according to the method further
comprises cholesterol. In particular embodiments, the DHSM and
cholesterol are present at a molar ratio from 75/25 (mol/mol)
DHSM/cholesterol to 25/75 (mol/mol) DHSM/cholesterol. In particular
embodiments, the DHSM and cholesterol are present at a molar ratio
from 60/40 (mol/mol) DHSM/cholesterol to 40/60 (mol/mol)
DHSM/cholesterol, or are present at a molar ratio of either about
55/45 (mol/mol) or about 50/50 (mol/mol) DHSM/cholesterol.
In a further embodiment, the liposomes used according to the method
comprise DHSM, cholesterol and other phospholipids or derivatized
phospholipids, wherein DHSM comprises at least 20% or at least 50%
of the total phospholipid present, and cholesterol is present at a
molar ratio from 75/25 (mol/mol) total phospholipid/cholesterol to
25/75 (mol/mol) total phospholipid/cholesterol. In particular
embodiments, the liposomes comprise DHSM, cholesterol and other
phospholipids or derivatized phospholipids, wherein DHSM comprises
at least 20% or at least 50% of the total phospholipid present, and
cholesterol is present at a molar ratio from 60/40 (mol/mol) total
phospholipid/cholesterol to 40/60 (mol/mol) total
phospholipid/cholesterol, and in further embodiments, total
phospholipid and cholesterol are present at either about 55/45
(mol/mol) total phospholipid/cholesterol or about 50/50 (mol/mol)
total phospholipid/cholesterol.
In related embodiments, the therapeutic agent used according to the
method of the invention is an antineoplastic agent. In particular
embodiments, the antineoplastic agent is one of the particular
agents described above.
In another related embodiment, the invention includes a method of
treating a cancer in a mammal, comprising administering to the
mammal a pharmaceutical composition comprising a
liposome-encapsulated therapeutic agent, wherein said liposome
comprises DHSM and wherein the DHSM comprises at least 20% or at
least 50% of the phospholipids present in said liposome. In a
particular embodiment, the liposome further comprises cholesterol.
In particular embodiments, the ratio of DHSM or total phospholipid
to cholesterol is any range or amount described herein.
In various embodiments, methods of the invention are used to treat
a variety of cancers, including a leukemia or lymphoma, or a solid
tumor, such as solid tumors of the lung, mammary, and colon. Such
treatments can be at first presentation of the cancer or in
patients who have relapsed after previous therapy.
The invention further provides a method of making a pharmaceutical
composition, comprising preparing a liposome comprising the
dihydrosphingomyelin and loading the prepared liposome with a
therapeutic compound.
In a related embodiment, the invention also provides a method of
manufacturing a pharmaceutical composition, comprising loading a
liposome comprising dihydrosphingomyelin with a therapeutic
compound.
In an additional embodiment, the invention includes a kit
comprising: a liposome comprising DHSM, wherein said DHSM comprises
at least 20% or at least 50% of the phospholipids present in said
liposome, and a therapeutic compound.
In a further related embodiment, the invention provides a method of
loading a therapeutic agent into a liposome, comprising: incubating
a liposome comprising DHSM and having an encapsulated medium
comprising MnSO.sub.4, wherein said DHSM comprises at least 20% or
at least 50% of the total phospholipids of the liposome, with an
external solution comprising said therapeutic agent and an
ionophore at a temperature greater than 60.degree. C. to form a
therapeutic agent-loaded liposome. In particular embodiments,
MnSO.sub.4 is present at a concentration equal to or greater than
300 mM. In one embodiment, MnSO.sub.4 is present at a concentration
of 600 mM. In related embodiments, the temperature at which the
therapeutic agent is loaded into the liposomes is less than or
equal to 70.degree. C. In a specific embodiment, the temperature is
70.degree. C.
In yet a further related embodiment, the present invention includes
a liposome comprising DHSM, wherein said DHSM constitutes at least
20% or at least 50% (molar basis) of total phospholipid present in
said liposome, and wherein the interior of said liposome comprises
MnSO.sub.4. In particular embodiments, the liposome may further
comprise an active agent. In one embodiment, the active agent is
topotecan.
In another embodiment, the present invention provides a composition
comprising empty liposomes and liposomes containing an active
agent, wherein said liposomes comprising an active agent comprise
MnSO.sub.4 and DHSM, wherein said DHSM constitutes at least 20% or
at least 50% (molar basis) of total phospholipid present in said
liposomes. In one embodiment, the active agent is topotecan.
In further related embodiments, the invention includes methods of
using a liposomal composition of the present invention to treat a
disease, e.g., tumor, by administering said liposomal composition
to a patient in need thereof.
The invention further includes kits comprising a liposome or
liposomal composition of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts concentration-normalized differential scanning
calorimetry (DSC) data for egg sphingomyelin (ESM) (A) or egg
dihydrosphingomyelin (EDHSM) (B). Samples were scanned from 20 to
60.degree. C. at a scan rate of 5.degree. C./hr.
FIG. 2 depicts concentration-normalized DSC data for milk
sphingomyelin (MSM) (A) or milk dihydrosphingomyelin (MDHSM) (B).
Samples were scanned from 20 to 60.degree. C. at a scan rate of
5.degree. C./hr.
FIG. 3 depicts concentration-normalized DSC data for brain
sphingomyelin (BSM) (A) or brain dihydrosphingomyelin (BDHSM) (B).
Samples were scanned from 20 to 60.degree. C. at a scan rate of
5.degree. C./hr.
FIG. 4 graphically depicts the uptake kinetics of topotecan into
various liposomal formulations. The formulations shown are ESM:Chol
Mg.sup.2+ (.circle-solid.); ESM Mn.sup.2+ (.largecircle.);
DHSM:Chol Mg.sup.2+ (.tangle-solidup.); and DHSM:Chol Mn.sup.2+
().
FIG. 5 depicts vincristine release from various liposome
compositions on incubation in 50% FBS at 50.degree. C. Liposome
formulations include DHSM:Chol (.box-solid.); ESM:Chol
(.circle-solid.); MSM:Chol (.tangle-solidup.); and BSM:Chol ().
FIG. 6 depicts vinorelbine release from various liposome
compositions on incubation in IVR release buffer at 37.degree. C.
The liposome compositions shown are ESM:Chol (.circle-solid.);
MDHSM:Chol (.box-solid.); BDHSM:Chol (.quadrature.); and EDHSM:Chol
(.largecircle.).
FIG. 7 depicts vinorelbine release from EPC:Chol liposomes
(.circle-solid.) and EPC:DHSM:Chol liposomes (.largecircle.) on
incubation in IVR release buffer at 25.degree. C.
FIG. 8 depicts the in vivo plasma drug retention associated with
liposomes comprising egg sphingomyelin (ESM) or egg
dihydrosphingomyelin (HESM). FIG. 8A illustrates vincristine
retention; FIG. 8B depicts NK611 retention; and FIG. 8C provides
topotecan retention.
FIGS. 9A-F provides the pharmacokinetic properties of various
liposomal topotecan formulations injected IV into ICR mice at 50 mg
lipid/kg. Figure A-C depict recovery from plasma, while Figures D-F
depict recovery from blood. Figures A and D depict drug retention
over time; Figures B and E depict lipid recovery over time; and
Figures C and F depict topotecan recovery over time. Data represent
the average of four mice.+-.one S.D. The liposomal compositions
shown are; ESM:Chol Mg.sup.2+ (.diamond-solid.); DHSM:Chol
Mg.sup.2+ (.box-solid.); ESM:Chol Mn.sup.2+ (.tangle-solidup.); and
DHSM:Chol Mn.sup.2+ (X).
FIG. 10 depicts plasma levels of ESM:Chol or DHSM:Chol liposomes at
various times after intravenous injection for two lipid dose
levels: (A) 25 mg/m.sup.2 and (B) 250 mg/m.sup.2.
FIG. 11 provides a graphical representation of the antitumor
activity of ESM/Chol and DHSM/Chol liposomal topotecan formulations
in MX-1 xenografts. All doses indicated were administered i.v.
q7d.times.3. The symbols represent: saline control
(.diamond-solid.); ESM/Chol/Mg.sup.2+, 1.0 mg/kg (.largecircle.);
ESM/Chol/Mg.sup.2+, 0.5 mg/kg (.quadrature.); DHSM/Chol/Mg.sup.2+,
1.0 mg/kg (.DELTA.); DHSM/Chol/Mg.sup.2+, 0.5 mg/kg (.gradient.);
ESM/Chol/Mn.sup.2+1.0 mg/kg (.circle-solid.); ESM/Chol/Mn.sup.2+,
0.5 mg/kg (.box-solid.); DHSM/Chol/Mn.sup.2+, 1.0 mg/kg (A); and
DHSM/Chol/Mn.sup.2+, 0.5 mg/kg (V). Data points represent median
tumor volumes (n=8). For graphical purposes, measured tumor volumes
below 63 mm.sup.3, the NCI defined limit of measurability, were
cut-off at 40 mm.sup.3.
FIG. 12 depicts treatment-related changes in body weights in the
MX-1 study. The percentage change in body weight was monitored
during the dosing phase (i.v. q7d.times.3) of the MX-1 study. The
symbols represent: saline control (.diamond-solid.);
ESM/Chol/Mg.sup.2+, 1.0 mg/kg (.largecircle.); ESM/Chol/Mg.sup.2+,
0.5 mg/kg (.quadrature.); DHSM/Chol/Mg.sup.2+, 1.0 mg/kg (.DELTA.);
DHSM/Chol/Mg.sup.2+, 0.5 mg/kg (.gradient.); ESM/Chol/Mn.sup.2+ 1.0
mg/kg (.circle-solid.); ESM/Chol/Mn.sup.2+, 0.5 mg/kg
(.box-solid.); DHSM/Chol/Mn.sup.2+, 1.0 mg/kg (.tangle-solidup.);
and DHSM/Chol/Mn.sup.2+, 0.5 mg/kg (). Data points represent group
means for percentage change in body weight (n=8).
FIG. 13 provides a graphical representation of the antitumor
activity of ESM/Chol/Mg.sup.2+ and DHSM/Chol/Mn.sup.2+ liposomal
topotecan formulations in HT-29 xenografts. All doses listed were
administered i.v. q4d.times.3. The symbols represent: saline
control (.diamond-solid.); ESM/Chol/Mg.sup.2+, 4.0 mg/kg
(.largecircle.); ESM/Chol/Mg.sup.2+, 2.0 mg/kg (.quadrature.);
ESM/Chol/Mg.sup.2+, 1.0 mg/kg (.DELTA.); ESM/Chol/Mg.sup.2+, 0.5
mg/kg (.gradient.); DHSM/Chol/Mn.sup.2+, 4 mg/kg (.circle-solid.);
DHSM/Chol/Mn.sup.2+, 2.0 mg/kg (.box-solid.); DHSM/Chol/Mn.sup.2+,
1.0 mg/kg (.tangle-solidup.); and DHSM/Chol/Mn.sup.2+, 0.5 mg/kg
(). Data points represent median tumor volumes (n=5). For graphical
purposes, measured tumor volumes below 63 mm.sup.3, the NCI defined
limit of measurability, were cut-off at 40 mm.sup.3.
FIG. 14 depicts treatment-related changes in body weights in the
HT-29 study. The percentage change in body weight was monitored
during the dosing phase (i.v. q4d.times.3) of the HT-29 study. The
symbols represent: saline control (.diamond-solid.); ESM/Chol/Mg2+,
4.0 mg/kg (.largecircle.); ESM/Chol/Mg2+, 2.0 mg/kg (.quadrature.);
ESM/Chol/Mg2+, 1.0 mg/kg (.DELTA.); ESM/Chol/Mg2+, 0.5 mg/kg
(.gradient.); DHSM/Chol/Mn2+4 mg/kg (.circle-solid.);
DHSM/Chol/Mn2+, 2.0 mg/kg (.box-solid.); DHSM/Chol/Mn2+, 1.0 mg/kg
(.tangle-solidup.); and DHSM/Chol/Mn2+, 0.5 mg/kg (). Data points
represent group means for percentage change in body weight
(.+-.standard deviation; n=5).
DETAILED DESCRIPTION OF THE INVENTION
The present invention includes novel liposomes and liposomal
compositions comprising therapeutic agents, as well as methods of
preparing and using such liposomes and liposomal compositions to
deliver therapeutic agents and treat diseases, including cancer.
The invention is fundamentally based on the surprising discovery
that liposomes comprising dihydrosphingomyelin (DHSM) have markedly
altered properties as compared to liposomes comprising
sphingomyelin (SM) and/or other phospholipids. Most notably,
liposomes prepared using DHSM show significantly increased
retention of active agents encapsulated within the liposomes, both
in vitro and in vivo, as compared to liposomes prepared using SM
and/or other phospholipids. This finding was entirely unexpected
given the structural characteristics and physical properties of SM
and DHSM, including those summarized below. In addition, liposomes
comprising DHSM exhibit longer plasma circulation half-lives
compared to similar liposomes comprising SM. Associated with these
changes in pharmacokinetics, liposomes comprising DHSM and
topotecan were found to exhibit greater antitumor activity compared
to similar liposomes comprised of SM in murine models of human
tumors.
Sphingomyelin comprises two variable components: the sphingosine
base (long chain base) and the long chain N-acyl residue (fatty
acid chain). The three main structures of long chain bases present
in sphingomyelin include: 4-sphingenine (sphingosine); sphinganine,
or, in its trivial name, dihydrosphingosine; and 4-D-hydroxy
sphingosine (phytosphingosine). Sphingosine
(trans-D-erythro-1,3-dihydroxy-2-amino-4-octadecene), also
described as trans-2S,3R)-2-amino-4-octadecene-1,3-diol, is the
main long chain base found in mammals; dihydrosphingosine
(trans-D-erythro, 1,3-dihydroxy-2-amino-4-octadecane or
(2S,3R)-2-amino-octadecane-1,3-diol) and phytosphingosine
(1,3,4-trihydroxy-D-ribo-2-amino-(2S,3R,4R)-2-amino-octadenane-1,3,4-trio-
l) are also found in eukaryotic sphingomyelin, but generally to a
much lesser extent. For example, in cultured mammalian cells, only
5-10% of sphingomyelin contains the dihydrosphingosine base
(Ramstedt et al. European Journal of Biochemistry, 266: 997-1002
(1999)). An exception is the human lens membranes where
dihydrosphingomyelin accounts for about 50% of the phospholipid
present (Byrdwell and Borchman, Ophthalmic Research, 29: 191-206
(1997)). Sphingosine comprises a trans double bond between carbons
4 and 5 of the sphingosine chain, whereas dihydrosphingosine lacks
this trans double bond. Representative examples of structures of
sphingomyelins comprising sphingosine or dihydrosphingosine are as
follows:
##STR00001##
The N-acyl composition of sphingomyelin of most mammalian sources
is characterized by a relatively high content of long-chain
saturated or monounsaturated acyl chains and a low content of
polyunsaturated acyl chains. In most mammalian tissues, palmitic
acid (C16:0) is the prevalent fatty acid, followed in decreased
abundance by nervonic acid (24:1), lignoceric acid (24:0), and
behenic acid (C22:0) (Barenholz, Y., In Physiology of Membrane
Fluidity, Vol. 1. Shinitsky, M., ed. CRC Press, Boca Raton, Fla.
131-174 (1984)). Greater than 93% of egg sphingomyelin has a
saturated N-acyl chain, as compared to phosphatidylcholines (PCs),
which typically have 23-47% saturated chains (e.g., soy PC is 23%
saturated; egg PC is 45% saturated).
Detailed structural analysis of sphingomyelins derived from various
natural sources, including egg, milk, and brain sphingomyelin,
confirms that the N-acyl chain is largely saturated or
monounsaturated. In addition, the vast majority of naturally
occurring sphingomyelin comprises a monounsaturated long chain base
(e.g. sphingosine base). Specifically, the most common long chain
bases detected were 16:1, 17:1, 18:1 and 19:1, whereas the most
common N-acyl chains detected were 16:0, 22:0, 23:0, and 24:0
(Karlsson, A., et al., Journal of Mass Spectrometry 33:1192-1198
(1998)).
As used herein, the general term sphingomyelin (SM) includes SMs
having any long chain base or N-acyl chain, including those
described above. The term dihydrosphingomyelin (DHSM) refers to SMs
comprising a sphinganine (i.e., dihydrosphingosine) long chain base
and, therefore, lacking the trans double bond in the long chain
base. DHSM may contain one or more cis double bonds in the N-acyl
chain. In a preferred embodiment, DHSM contains both fully
saturated N-acyl chain and a saturated long base chain. In
addition, the term hydrogenated SM refers generally to SMs that
have been hydrogenated by any method available in the art.
Dihydrosphingomyelin is more specifically defined herein as any
N-acyl-sphinganyl-1-O-phosphorylcholine derivative. Sphinganine is
a natural product that typically is composed primarily of
D-erythro-2-amino-octadecane-1,3-diol, although material from some
sources may also contain significant amounts of
D-erythro-2-amino-heptadecane-1,3-diol. The sphinganine backbone of
dihydrosphingomyelin is defined here more generally to include any
D-erythro-2-amino-alkane-1,3-diol wherein the alkane is a linear
chain 12 to 24 carbon atoms in length, or any mixture thereof.
The presence of the trans double bond between carbons 4 and 5 in
the sphingosine base has been shown to impart specific structural
and physiological properties upon SMs. For example, analysis of the
monolayer properties of SM and DHSM showed that their packing
properties are very similar, except that the expanded-to-condensed
phase transition (crystallization) occurred at a lower pressure for
DHSM as compared to SM (Kuikka, M. et al., Biophysical Journal
80:2327-2337 at 2335 (2001)). Furthermore, it has been shown that
the surface potential of DHSM monolayers is reduced compared with
SM monolayers, possibly originating from an inducible dipole due to
the trans double bond being present in SM but absent in DHSM
(Kuikka, M. et al., at 2335 (2001)).
Interestingly, it was demonstrated that 16:0-DHSM was degraded much
faster by sphingomyelinase from Staphylococcus aureus than 16:0-SM,
and a ten-fold difference in enzyme activity was needed to produce
a comparable hydrolysis rate (Kuikka, M. et al., Biophysical
Journal 80:2327-2337 at 2330-2331 (2001)). The authors suspected
that packing heterogeneity (defects) similar to those seen at
boundaries between ordered and disordered membrane domains were
responsible for the increased susceptibility of DHSM to enzymatic
degradation. Without wishing to be bound by any particular theory,
it is noted that the increased susceptibility to enzymatic
degradation suggests that, upon administration to a patient,
liposomes comprising DHSM are susceptible to more rapid clearance
from the bloodstream and/or more rapid release of encapsulated
compounds, e.g., drugs, as compared to liposomes comprising SM
having the trans double bond.
The presence of the trans-double bond between carbon atoms 4 and 5
of the sphingenine moiety has been shown to have little effect on
the character of the gel-liquid crystalline phase transition of
SMs. For example, the difference between the Tm values for 16:0-SM
and 16:0-DHSM is only 6.5.degree. C. (Kuikka, M. et al.,
Biophysical Journal 80:2327-2337 at 2331-2333 (2001)). In
comparison, the difference in Tm between a phosphatidylcholine (PC)
possessing saturated (16:0) fatty acid chains and monounsaturated
(16:1) fatty acid chains is approximately 77.degree. C. (16:0 PC,
Tm=41.degree. C.; 16:1 PC, Tm=-36.degree. C.) (Marsh, D. CRC
Handbook of Lipid Bilayers, CRC Press, Boca Raton, Fla. (1990) at
p. 139 and p. 144). The relatively small effect on Tm value from
hydrogenation of the trans double bond in SM has been ascribed to
the position of this bond in the structurally ordered interface,
where it is not expected to influence considerably the packing
order of the hydrocarbon chains and thus effect the chain
order-disorder transition (Konova, R. and Caffrey, M., Biochim.
Biophys. Acta 1255:213-236 (1995).
Furthermore, the presence of the trans double-bond in DHSM does not
appear to affect its interaction with cholesterol in mixed
monolayers when cholesterol is present at 50 mol % or less. Studies
measuring cholesterol desorption from monolayers to cyclodextrin
acceptors in the subphase, which was used as a measure of how well
cholesterol interacts with other lipids in a mixed monolayer,
revealed that the desorption rate was practically zero using either
16:0-SM or 16:0-DHSM when the cholesterol concentration in the
mixed monolayer was 50 mol % (Kuikka, M. et al., Biophysical
Journal 80:2327-2337 (2001), p. 2330, col. 2, lines 8-29).
Based on these studies demonstrating little impact of the presence
or absence of the trans double bond on packing density or Tm, it
was extremely surprising to discover that hydrogenation of this
trans double bond resulted in liposomes having increased retention
of active agents encapsulated within. Furthermore, the magnitude of
the observed effect was also very surprising, given that
naturally-occurring sphingomyelin comprises only the single trans
double bond and generally either no cis double bonds or only one
cis double bond. Accordingly, it was surprising to discover that
liposomal compositions prepared from liposomes comprising DHSM and
a therapeutic agent provide unexpected advantages in drug delivery,
including both increased retention of the therapeutic agent in the
liposome in vitro, increased plasma drug retention in vivo, long
plasma circulation half-lives for both the liposomes and drug, and
increased antitumor activity against human tumor xenografts in a
murine model.
A. Liposomes Comprising Dihydrosphingomyelin
The present invention includes liposomes comprising DHSM or
hydrogenated SM. As used herein, a liposome is a structure having
lipid-containing membranes enclosing an aqueous interior. Liposomes
may have one or more lipid membranes. The invention contemplates
both single-layered liposomes, which are referred to as
unilamellar, and multi-layer liposomes, which are referred to as
multilamellar.
In various embodiments, the invention contemplates liposomes
comprising any naturally occurring or synthetically produced DHSM,
including those described in further detail infra. These liposomes
may further comprise one or more additional lipids and/or other
components such as cholesterol. Specific embodiments of liposomes
of the invention and their various components are described
below.
1. Sphingomyelin
The liposomes of the present invention comprise
dihydrosphingomyelin (DHSM) or hydrogenated sphingomyelin,
including, but not limited to, any naturally occurring,
semi-synthetic or synthetic DHSM described herein.
Naturally occurring SMs have the phosphocholine head group linked
to the hydroxyl group on carbon one of a long-chain base and have a
long and highly saturated acyl chain linked to the amide group on
carbon 2 of the long-chain base (reviewed in Barenholz, Y. in
Physiology of Membrane Fluidity, Vol. 1. M. Shinitsky, editor. CRC
Press, Boca Raton, Fla. 131-174 (1984)). In cultured cells, about
90 to 95% of the SMs contain sphingosine
(1,3-dihydroxy-2-amino-4-octadecene), which contains a trans-double
bond between C4 and C5, as the long-chain base, whereas most of the
remainder have sphinganine (1,3-dihydroxy-2-amino-4-octadecane) as
the base and lack the trans double bond between carbons 4 and 5 of
the long chain base. The latter SMs are called
dihydrosphingomyelins (DHSM).
Other bases varying in length, degree of hydroxylation, and
branching are also found in nature. The enantiomeric configuration
of the sphingoid base in natural SMs is D-erythro (2S,3R).
Synthetically produced SMs may comprise either the D-erythro or
L-erythro configuration or a mixture of both.
Natural SMs usually constitute a mixed population with the
amide-linked acyl chain differing widely in length (generally from
16-24 carbons). The SM N-acyl chain composition varies between
tissues, although a common feature of naturally occurring SMs is
that the chains are usually long. Most tissues contain SMs with
16:0, 18:0, 22:0, 24:0 and 24:1.sup.cis.DELTA.15 N-acyl chains. In
SM, there is also a high frequency of saturated amide-linked acyl
chains with an average of only 0.1-0.35 cis-double bonds per
molecule. When present, the cis-double bond in natural SM is
typically located far away from the interface, as in nervonic acid
(24:1.sup.cis.DELTA.15) with a double bond at C.sub.15. The
interfacial region of SM has an amide group, a free hydroxyl on
C.sub.3 and the trans-double bond between C.sub.4 and C.sub.5 in
this region.
The N-acyl composition of SM isolated from natural sources is
provided in Table 1. Reference in Table 1 to saturated or
unsaturated is specific for the fatty acid chain of sphingomyelin
derived from the various sources and does not indicate the presence
or absence of the trans double bond in the long chain base. For
consistency with the nomenclature used with other phospholipids,
such as phosphatidylcholine, the N-acyl chains on SM are sometimes
referred to as fatty acids. However it is to be understood that
these acyl chains are linked to the sphingosine base via an amide
bond and not via an ester bond such as is present with most other
phospholipids.
TABLE-US-00001 TABLE 1 N-Acyl chain compositions of sphingomyelin
(wt % of the total) from various sources Tissue Derived N-Acyl
Chain Egg Egg Brain Brain Milk Milk Composition SM.sup.1 SM.sup.2
SM.sup.1 SM.sup.2 SM.sup.1 SM.sup.2 SATURATED 16:0 84% 66% 2% 3%
19% 14% 18:0 6% 10% 46% 42% 3% 3% 20:0 2% 4% 5% 6% 1% 1% 22:0 4% 6%
7% 7% 19% 22% 23:0 2% 3% 33% 32% 24:0 4% 5% 6% 20% 19% SUBTOTAL
100% 93% 60% 67% 95% 91% UNSATURATED 18:1 1% 1% 20:4 2% 22:1 1% 3%
23:1 3% 24:1 3% 6% 27% 3% 5% SUBTOTAL 5% 6% 33% 5% 6% UNKNOWN Other
34% TOTAL 100% 98% 100% 100% 100% 97% .sup.1Avanti - based on Wood
& Holton (1964) Proc. Soc. Exptl. Biol. Med 115, 990
.sup.2based on Ramstedt B, Leppimaki P, Axberg M, Slotte JP (1999)
Analysis of natural and synthetic sphingomyelins using
high-performance thin-layer chromatography. Eur J Biochem. 266(3):
997-1002
The present invention includes liposomes comprising DHSM having
N-acyl or fatty acid chains of any length, including, e.g., 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24
carbon atoms. In certain embodiments, the N-acyl chain consists of
12 to 24 carbon atoms, including 12 or 24 carbon atoms and any
integer value within this range. In specific embodiments, the acyl
chain consists of 12, 16 or 18 carbon atoms, and in one specific
embodiment, it consists of 16 carbon atoms. In another embodiment,
it consists of 18 carbon atoms. In another embodiment, the
invention includes liposomes having DHSM species having the above
chain lengths.
In one embodiment, the invention includes DHSM having matched chain
lengths. In naturally occurring SMs, the two chains, i.e., the
N-acyl chain and the acyl chain contributed by the sphingosine
base, are roughly of equal length (matched) when the N-acyl chain
is about 16 carbons long, since the sphingosine acyl chain is
mostly of constant length in all molecular species (see above
discussion of variations in sphingosine acyl length). Thus, in
certain embodiments directed to matched chains DHSM, the N-acyl
chain is about 16 carbons long, 16-18 carbons long, or 16 carbons
long. In a related embodiment, the N-acyl chain and the sphingosine
acyl chain consist of carbon chains not different in length by more
than four carbon atoms. In another embodiment, the invention
includes liposomes having hydrogenated sphingomyelin having matched
chain lengths.
SM isolated from various sources is commercially available (Avanti
Polar Lipids, Alabaster, Ala.), and DHSM may be prepared from SM by
hydrogenation by any means available in the art. Hydrogenation
procedures that may be used according to the invention include,
e.g., those described in Kuikka, M. et al., Biophys. J. 80:2327-37
(2001) and references cited therein; Barenholz, Y., et al.,
Biochemistry 15(11):2441-2447 (1976) and references cited therein;
and Ollila, F. and Slotte, J. P., Biochim. Biophys. Acta
1564:281-288 (2002) and references cited therein. Alternatively,
DHSM could be prepared synthetically starting, for example, with
dihydrosphingosine, by any means available in the art.
The majority of SMs lack any cis double bonds (see Table 1). Thus,
hydrogenation of SM generally targets the trans double bond of the
sphingosine base, resulting in DHSM. Of course, hydrogenation of SM
would likely also result in hydrogenation of any cis double bonds
present in the N-acyl chain. However, it should be understood that
according to the present invention, DHSM may comprise one or more
cis double bonds in the acyl chain, so long as the trans double
bond of the sphingosine base is absent. In one particular
embodiment, however, DHSMs of the present invention lack any cis
double bonds in the acyl chain and also lack the trans double bond
in the sphingosine base.
In certain embodiments of liposomes of the present invention, and
the related methods of the present invention, at least 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100% (molar basis)
of the total phospholipids present in the liposome are DHSM. In one
particular embodiment, DHSM comprises at least 50% (molar basis) of
the total phospholipids present in the liposome. In another
embodiment, DHSM comprises at least 20% (molar basis) of the total
phospholipids present in the liposome.
Liposomes comprising DHSM may also further comprise SM that
contains the trans double bond in the sphingosine base.
Accordingly, in certain embodiments, at least 10%, at least 20%, at
least 30%, at least 40%, at least 50%, at least 60%, at least 70%,
at least 80%, at least 90%, at least 95%, at least 99%, or 100%
(molar basis) of the total SM present in a liposome of the
invention is DHSM. In one preferred embodiment, at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, or 100% (molar
basis) of the SM present in a liposome of the invention is
DHSM.
2. Other Lipids and Liposome Components
Liposomes of the invention may further comprise additional lipids
and other components. Other lipids may be included in the liposome
compositions of the present invention for a variety of purposes,
such as to prevent lipid oxidation or to attach ligands onto the
liposome surface. Any of a number of lipids may be present in
liposomes of the present invention, including amphipathic, neutral,
cationic, and anionic lipids. Such lipids can be used alone or in
combination, and can also include bilayer stabilizing components
such as polyamide oligomers (see, e.g., U.S. Pat. No. 6,320,017),
peptides, proteins, detergents, lipid-derivatives, such as PEG
coupled to phosphatidylethanolamine and PEG conjugated to ceramides
(see, U.S. Pat. No. 5,885,613).
Any of a number of neutral lipids can be included, referring to any
of a number of lipid species that exist either in an uncharged or
neutral zwitterionic form at physiological pH, including, e.g.,
diacylphosphatidylcholine, diacylphosphatidylethanolamine,
ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides,
diacylglycerols, and sterols.
In certain embodiments, the liposomes of the present invention
comprises DHSM and cholesterol. Liposomes comprising SM and
cholesterol are referred to as sphingosomes and are described in
U.S. Pat. Nos. 5,543,152, 5,741,516, and 5,814,335. The ratio of
DHSM to cholesterol in the liposome composition can vary, but
generally is in the range of from about 75/25 (mol/mol)
DHSM/cholesterol to about 25/75 (mol/mol) DHSM/cholesterol, more
preferably about 60/40 (mol/mol) DHSM/cholesterol to about 40/60
(mol/mol) DHSM/cholesterol, and even more preferably about 55/45
(mol/mol) or 50/50 (mol/mol) DHSM/cholesterol. Generally, if other
lipids are included, the inclusion of such lipids will result in a
decrease in the DHSM/cholesterol ratio.
In certain embodiments, the liposomes of the present invention
comprises DHSM and cholesterol, as well as one or more other
phospholipids. The ratio of total phospholipid to cholesterol in
the liposome composition can vary, but generally is in the range of
from about 75/25 (mol/mol) total phospholipid/cholesterol to about
25/75 (mol/mol) total phospholipid/cholesterol, from about 60/40
(mol/mol) total phospholipid/cholesterol to about 40/60 (mol/mol)
total phospholipid/cholesterol, or about 55/45 (mol/mol) or 50/50
(mol/mol) total phospholipid/cholesterol.
Cationic lipids, which carry a net positive charge at about
physiological pH, can readily be incorporated into liposomes for
use in the present invention. Such lipids include, but are not
limited to, N,N-dioleyl-N,N-dimethylammonium chloride ("DODAC");
N-(2,3-dioleyloxy)propyl-N,N-N-triethylammonium chloride ("DOTMA");
N,N-distearyl-N,N-dimethylammonium bromide ("DDAB");
N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
("DOTAP");
3.beta.-(N--(N',N'-dimethylaminoethane)-carbamoyl)cholesterol
("DC-Chol"),
N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-
ammonium trifluoracetate ("DOSPA"), dioctadecylamidoglycyl
carboxyspermine ("DOGS"), 1,2-dileoyl-sn-3-phosphoethanolamine
("DOPE"), 1,2-dioleoyl-3-dimethylammonium propane ("DODAP"),
N,N-dimethyl-2,3-dioleyloxy)propylamine ("DODMA"), and
N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium
bromide ("DMRIE"). Additionally, a number of commercial
preparations of cationic lipids can be used, such as, e.g.,
LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL),
and LIPOFECTAMINE (comprising DOSPA and DOPE, available from
GIBCO/BRL).
Anionic lipids suitable for use in the present invention include,
but are not limited to, phosphatidylglycerol, cardiolipin,
diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoyl
phosphatidylethanoloamine, N-succinyl phosphatidylethanolamine,
N-glutaryl phosphatidylethanolamine, lysylphosphatidylglycerol, and
other anionic modifying groups joined to neutral lipids.
In numerous embodiments, amphipathic lipids are included in
liposomes of the present invention. "Amphipathic lipids" refer to
any suitable material, wherein the hydrophobic portion of the lipid
material orients into a hydrophobic phase, while the hydrophilic
portion orients toward the aqueous phase. Such compounds include,
but are not limited to, phospholipids, aminolipids, and
sphingolipids. Representative phospholipids include sphingomyelin,
phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, phosphatidic acid, palmitoyloleoyl
phosphatdylcholine, lysophosphatidylcholine,
lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,
dioleoylphosphatidylcholine, distearoylphosphatidylcholine, or
dilinoleoylphosphatidylcholine. Other phosphorus-lacking compounds,
such as sphingolipids, glycosphingolipid families, diacylglycerols,
and .beta.-acyloxyacids, can also be used. Additionally, such
amphipathic lipids can be readily mixed with other lipids, such as
triglycerides and sterols.
In one embodiment, cloaking agents, which reduce elimination of
liposomes by the host immune system, can also be included in
liposomes of the present invention, such as polyamide-oligomer
conjugates, e.g., ATTA-lipids, (see, U.S. Pat. No. 6,320,017) and
PEG-lipid conjugates (see, U.S. Pat. Nos. 5,820,873, 5,534,499 and
5,885,613).
Also suitable for inclusion in the present invention are
programmable fusion lipid formulations. Such formulations have
little tendency to fuse with cell membranes and deliver their
payload until a given signal event occurs. This allows the lipid
formulation to distribute more evenly after injection into an
organism or disease site before it starts fusing with cells. The
signal event can be, for example, a change in pH, temperature,
ionic environment, or time. In the latter case, a fusion delaying
or "cloaking" component, such as an ATTA-lipid conjugate or a
PEG-lipid conjugate, can simply exchange out of the liposome
membrane over time. By the time the formulation is suitably
distributed in the body, it has lost sufficient cloaking agent so
as to be fusogenic. With other signal events, it is desirable to
choose a signal that is associated with the disease site or target
cell, such as increased temperature at a site of inflammation.
In certain embodiments, it is desirable to target the liposomes of
this invention using targeting moieties that are specific to a cell
type or tissue. Targeting of liposomes using a variety of targeting
moieties, such as ligands, cell surface receptors, glycoproteins,
vitamins (e.g., riboflavin) and monoclonal antibodies, has been
previously described (see, e.g., U.S. Pat. Nos. 4,957,773 and
4,603,044). The targeting moieties can comprise the entire protein
or fragments thereof.
Targeting mechanisms generally require that the targeting agents be
positioned on the surface of the liposome in such a manner that the
target moiety is available for interaction with the target, for
example, a cell surface receptor. A variety of different targeting
agents and methods are known and available in the art, including
those described, e.g., in Sapra, P. and Allen, T M, Prog. Lipid
Res. 42(5):439-62 (2003); and Abra, R M et al., J. Liposome Res.
12:1-3, (2002).
The use of liposomes with a surface coating of hydrophilic polymer
chains, such as polyethylene glycol (PEG) chains, for targeting has
been proposed (Allen, et al., Biochimica et Biophysica Acta 1237:
99-108 (1995); DeFrees, et al., Journal of the American Chemistry
Society 118: 6101-6104 (1996); Blume, et al., Biochimica et
Biophysica Acta 1149: 180-184 (1993); Klibanov, et al., Journal of
Liposome Research 2: 321-334 (1992); U.S. Pat. No. 5,013,556;
Zalipsky, Bioconjugate Chemistry 4: 296-299 (1993); Zalipsky, FEBS
Letters 353: 71-74 (1994); Zalipsky, in Stealth Liposomes Chapter 9
(Lasic and Martin, Eds) CRC Press, Boca Raton Fla. (1995). In one
approach, a ligand, such as an antibody, for targeting the
liposomes is linked to the polar head group of lipids forming the
liposome. In another approach, the targeting ligand is attached to
the distal ends of the PEG chains forming the hydrophilic polymer
coating (Klibanov, et al., Journal of Liposome Research 2: 321-334
(1992); Kirpotin et al., FEBS Letters 388: 115-118 (1996)).
In one embodiment, the liposome is designed to incorporate a
connector portion into the membrane at the time of liposome
formation. The connector portion must have a lipophilic portion
that is firmly embedded and anchored into the membrane. It must
also have a hydrophilic portion that is chemically available on the
aqueous surface of the liposome. The hydrophilic portion is
selected so as to be chemically suitable with the targeting agent,
such that the portion and agent form a stable chemical bond.
Therefore, the connector portion usually extends out from the
liposomal surface and is configured to correctly position the
targeting agent. In some cases, it is possible to attach the target
agent directly to the connector portion, but in many instances, it
is more suitable to use a third molecule to act as a "molecular
bridge." The bridge links the connector portion and the target
agent off of the surface of the liposome, thereby making the target
agent freely available for interaction with the cellular
target.
Standard methods for coupling the target agents can be used. For
example, phosphatidylethanolamine, which can be activated for
attachment of target agents, or derivatized lipophilic compounds,
such as lipid-derivatized bleomycin, can be used. Antibody-targeted
liposomes can be constructed using, for instance, liposomes that
incorporate protein A (see, Renneisen, et al., J. Bio. Chem.,
265:16337-16342 (1990) and Leonetti, et al., Proc. Natl. Acad. Sci.
(USA), 87:2448-2451 (1990). Other examples of antibody conjugation
are disclosed in U.S. Pat. No. 6,027,726, the teachings of which
are incorporated herein by reference. Examples of targeting
moieties can also include other proteins, specific to cellular
components, including antigens associated with neoplasms or tumors.
Proteins used as targeting moieties can be attached to the
liposomes via covalent bonds (see, Heath, Covalent Attachment of
Proteins to Liposomes, 149 Methods in Enzymology 111-119 (Academic
Press, Inc. 1987)). Other targeting methods include the
biotin-avidin system.
3. Methods of Preparing Liposomes
A variety of methods for preparing liposomes are known in the art,
including e.g., those described in Szoka, et al., Ann. Rev.
Biophys. Bioeng., 9:467 (1980); U.S. Pat. Nos. 4,186,183,
4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085,
4,837,028, 4,946,787; PCT Publication No. WO 91/17424; Deamer and
Bangham, Biochim. Biophys. Acta, 443:629-634 (1976); Fraley, et
al., Proc. Natl. Acad. Sci. USA, 76:3348-3352 (1979); Hope, et al.,
Biochim. Biophys. Acta, 812:55-65 (1985); Mayer, et al., Biochim.
Biophys. Acta, 858:161-168 (1986); Williams, et al., Proc. Natl.
Acad. Sci., 85:242-246 (1988); Liposomes, Marc J. Ostro, ed.,
Marcel Dekker, Inc., New York, 1983, Chapter 1; Hope, et al., Chem.
Phys. Lip., 40:89 (1986); and Liposomes: A Practical Approach,
Torchilin, V. P. et al., ed., Oxford University Press (2003), and
references cited therein. Suitable methods include, but are not
limited to, sonication, extrusion, high pressure/homogenization,
microfluidization, detergent dialysis, calcium-induced fusion of
small liposome vesicles, and ether-infusion methods, all of which
are well known in the art.
Alternative methods of preparing liposomes are also available. For
instance, a method involving detergent dialysis based self-assembly
of lipid particles is disclosed and claimed in U.S. Pat. No.
5,976,567, which avoids the time-consuming and difficult to-scale
drying and reconstitution steps.
One method produces multilamellar vesicles of heterogeneous sizes
(Bangham, A. and Haydon, D. A., Br Med Bull. 24(2):124-6 (1968) and
Bangham, A. D., Prog Biophys Mol. Biol. 18:29-95 (1968)). In this
method, the vesicle-forming lipids are dissolved in a suitable
organic solvent or solvent system and dried under vacuum or an
inert gas to form a thin lipid film. If desired, the film may be
redissolved in a suitable solvent, such as tertiary butanol, and
then lyophilized to form a more homogeneous lipid mixture which is
in a more easily hydrated powder-like form. This film is covered
with an aqueous buffered solution and allowed to hydrate, typically
over a 15-60 minute period with agitation. The size distribution of
the resulting multilamellar vesicles can be shifted toward smaller
sizes by hydrating the lipids under more vigorous agitation
conditions or by adding solubilizing detergents, such as
deoxycholate. Multilamellar vesicles can also be made by dissolving
phospholipids in ethanol and then injecting the ethanol solution
into a buffer, causing the lipids to spontaneously form liposomes.
Further, the trapped volume of the multilamellar vesicles can be
increased by a freeze-thaw procedure (Mayer, L D et al., Biochim.
Biophys. Acta, 817:193-196 (1985)).
Multilamellar vesicles formed on hydration of lipids in buffer are
generally heterogeneous in size and contain several concentric
lipid bilayers. In many applications, homogeneous liposomes
consisting predominantly of only a single bilayer (unilamellar
vesicles) and of a size range of about 100 nm to 200 nm are
preferred. Several techniques are available for sizing liposomes to
a desired size. General methods for sizing liposomes include, e.g.,
sonication, by bath or by probe, or homogenization, including the
method described in U.S. Pat. No. 4,737,323. Sonicating a liposome
suspension either by bath or probe sonication produces a
progressive size reduction down to small unilamellar vesicles less
than about 0.05 microns in size. Homogenization is another method
that relies on shearing energy to fragment large liposomes into
smaller ones. In a typical homogenization procedure, multilamellar
vesicles are recirculated through a standard emulsion homogenizer
until selected liposome sizes, typically between about 0.1 and 0.5
microns, are observed. The size of the liposomal vesicles may be
determined by quasi-electric light scattering (QELS) as described
in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-450 (1981),
incorporated herein by reference. Average liposome diameter may be
reduced by sonication of formed liposomes. Intermittent sonication
cycles may be alternated with QELS assessment to guide efficient
liposome synthesis.
Extrusion of liposome through a small-pore polycarbonate membrane
or an asymmetric ceramic membrane is also an effective method for
reducing liposome sizes to a relatively well-defined size
distribution. Typically, the suspension is cycled through the
membrane one or more times until the desired liposome size
distribution is achieved. The liposomes may be extruded through
successively smaller-pore membranes, to achieve gradual reduction
in liposome size. Liposome size can be determined and monitored by
known techniques, including, e.g., conventional laser-beam particle
size discrimination or the like. Extrusion may be carried out using
purpose-built extruders, such as the Lipex Extruder. Defined pore
size in the extrusion filters may generate unilamellar liposomal
vesicles of specific sizes (Hope M J et al. Biochim. Biophys. Acta,
812: 55-65 (1985)). The liposomes may also be formed by extrusion
through an asymmetric ceramic filter, such as a Ceraflow
Microfilter, commercially available from the Norton Company,
Worcester Mass.
Other methods of producing unilamellar vesicles are known. For
example, phospholipids can be solubilized into a detergent, e.g.,
cholates, Triton-X100, or n-alkylglucosides. Following removal of
the detergent by any of a number of possible methods including
dialysis and gel filtration, liposomes can be formed.
Liposomes of any size may be used according to the present
invention. In certain embodiments, liposomes of the present
invention have a size ranging from about 0.05 microns to about 0.45
microns, between about 0.05 and about 0.2 microns, or between 0.08
and 0.12 microns in diameter. In one embodiment, liposomes of the
present invention are about 0.1 microns in diameter. In other
embodiments, liposomes of the present invention are between about
0.45 microns to about 3.0 microns, about 1.0 to about 2.5 microns,
about 1.5 to about 2.5 microns and about 2.0 microns.
In certain embodiments, the liposomes used in the present invention
comprise a pH gradient across the membrane. In one embodiment, the
pH is lower at the interior of the liposomes than at the exterior.
Such gradients can be achieved, e.g., by formulating the liposomes
in the presence of a buffer with a low pH, e.g., having a pH
between about 2 and about 6, and subsequently transferring the
liposomes to a higher pH solution. For example, before or after
sizing of liposomes, the external pH can be raised, e.g., to about
7 or 7.5, by the addition of a suitable buffer, such as a sodium
phosphate buffer. Raising the external pH creates a pH gradient
across the liposomal membrane, which promotes efficient drug
loading and retention. In one embodiment, the internal pH is
between about 3 and 5, and in another embodiment, the internal pH
is about 4. Any of a number of buffers can be used, including,
e.g., acetate, tartrate, phosphate and citrate buffers.
In numerous embodiments, the liposomes are first formulated in a
low pH buffer, and then manipulated in one of a variety of ways to
obtain liposomes of the desired size. A pH gradient is then formed
by transferring the liposomes into a medium of higher pH or by
increasing the pH of the external medium.
In one embodiment, the liposomes used in the present invention
comprise a transmembrane potential, while in another embodiment,
liposomes of the invention do not comprise a transmembrane
potential.
Liposomes prepared according to these methods can be stored for
substantial periods of time prior to drug loading and
administration to a patient. For example, liposomes can be
dehydrated, stored, and subsequently rehydrated and loaded with one
or more active agents, prior to administration. Liposomes may also
be dehydrated after being loaded with one or more active agents.
Dehydration can be accomplished by a variety of methods available
in the art, including the dehydration and lyophilization procedures
described, e.g., in U.S. Pat. Nos. 4,880,635, 5,578,320, 5,837,279,
5,922,350, 4,857,319, 5,376,380, 5,817,334, 6,355,267, and
6,475,517. In one embodiment, liposomes are dehydrated using
standard freeze-drying apparatus, i.e., they are dehydrated under
low pressure conditions. Also, the liposomes can be frozen, e.g.,
in liquid nitrogen, prior to dehydration. Sugars can be added to
the liposomal environment, e.g., to the buffer containing the
liposomes, prior to dehydration, thereby promoting the integrity of
the liposome during dehydration. See, e.g., U.S. Pat. No. 5,077,056
or 5,736,155.
Liposomes may be sterilized by conventional methods at any point
during their preparation, including, e.g., after sizing or after
generating a pH gradient.
B. Liposomal Compositions Comprising Active Agents
In various embodiments, liposomes of the present invention may be
used for many different applications, including the delivery of an
active agent to a cell, tissue, organ or subject. For example,
liposomes of the invention may be used to deliver a therapeutic
agent systemically via the bloodstream or to deliver a cosmetic
agent to the skin. Accordingly, liposomal compositions comprising a
liposome of the present invention and one or more active agents are
included in the present invention.
1. Active Agents
The present invention includes liposomal compositions comprising a
liposome of the present invention (i.e., a liposome comprising
DHSM) and an active agent. Active agents, as used herein, include
any molecule or compound capable of exerting a desired effect on a
cell, tissue, organ, or subject. Such effects may be biological,
physiological, or cosmetic, for example. Active agents may be any
type of molecule or compound, including e.g., nucleic acids, such
as single- or double-stranded polynucleotides, plasmids, antisense
RNA, RNA interference reagents, including, e.g., DNA-DNA hybrids,
DNA-RNA hybrids, RNA-DNA hybrids, RNA-RNA hybrids, short
interfering RNAs (siRNA), micro RNAs (mRNA) and short hairpin RNAs
(shRNAs); peptides and polypeptides, including, e.g., antibodies,
such as, e.g., polyclonal antibodies, monoclonal antibodies,
antibody fragments; humanized antibodies, recombinant antibodies,
recombinant human antibodies, and Primatized.TM. antibodies,
cytokines, growth factors, apoptotic factors,
differentiation-inducing factors, cell surface receptors and their
ligands; hormones; and small molecules, including small organic
molecules or compounds.
In one embodiment, the active agent is a therapeutic agent, or a
salt or derivative thereof. Therapeutic agent derivatives may be
therapeutically active themselves or they may be prodrugs, which
become active upon further modification. Thus, in one embodiment, a
therapeutic agent derivative retains some or all of the therapeutic
activity as compared to the unmodified agent, while in another
embodiment, a therapeutic agent derivative lacks therapeutic
activity.
In various embodiments, therapeutic agents include many agents and
drugs, such as anti-inflammatory compounds, narcotics, depressants,
anti-depressants, stimulants, hallucinogens, analgesics,
antibiotics, birth control medication, antipyretics, vasodilators,
anti-angiogenics, cytovascular agents, signal transduction
inhibitors, vasoconstrictors, hormones, and steroids.
In certain embodiments, the active agent is an oncology drug, which
may also be referred to as an anti-tumor drug, an anti-cancer drug,
a tumor drug, an antineoplastic agent, or the like. Examples of
oncology drugs that may be used according to the invention include,
but are not limited to, adriamycin, alkeran, allopurinol,
altretamine, amifostine, anastrozole, araC, arsenic trioxide,
azathioprine, bexarotene, biCNU, bleomycin, busulfan intravenous,
busulfan oral, capecitabine (Xeloda), carboplatin, carmustine,
CCNU, celecoxib, chlorambucil, cisplatin, cladribine, cyclosporin
A, cytarabine, cytosine arabinoside, daunorubicin, cytoxan,
daunorubicin, dexamethasone, dexrazoxane, dodetaxel, doxorubicin,
doxorubicin, DTIC, epirubicin, estramustine, etoposide phosphate,
etoposide and VP-16, exemestane, FK506, fludarabine, fluorouracil,
5-FU, gemcitabine (Gemzar), gemtuzumab-ozogamicin, goserelin
acetate, hydrea, hydroxyurea, idarubicin, ifosfamide, imatinib
mesylate, interferon, irinotecan (Camptostar, CPT-111), letrozole,
leucovorin, leustatin, leuprolide, levamisole, litretinoin,
megastrol, melphalan, L-PAM, mesna, methotrexate, methoxsalen,
mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel,
pamidronate, Pegademase, pentostatin, porfimer sodium, prednisone,
rituxan, streptozocin, STI-571, tamoxifen, taxotere, temozolamide,
teniposide, VM-26, topotecan (Hycamtin), toremifene, tretinoin,
ATRA, valrubicin, velban, vinblastine, vincristine, VP16, and
vinorelbine. Other examples of oncology drugs that may be used
according to the invention are ellipticin and ellipticin analogs or
derivatives, epothilones, intracellular kinase inhibitors and
camptothecins.
In one embodiment, liposomes of the present invention are used to
deliver an alkaloid. Accordingly, the invention includes liposomal
compositions comprising one or more alkaloids. The present
invention includes any naturally occurring alkaloid, including
vinca alkaloids, or any synthetic derivative of a naturally
occurring alkaloid. Vinca alkaloids include, but are not limited
to, vinblastine, vincristine, vindoline, vindesine, vinleurosine,
vinrosidine, vinorelbine, or derivatives thereof (see, e.g., the
Merck Index, 11.sup.th Edition (1989) entries 9887, 9891, and 9893,
for vinblastine, vincristine, and vindoline).
In another embodiment, liposomes of the present invention are used
to deliver podophyllins, podophyllotoxins, and derivatives thereof
(e.g., etoposide, etoposide phosphate, teniposide, etc.),
camptothecins (e.g., irinotecan, topotecan, etc.), and taxanes
(paclitaxol, etc.), and derivatives thereof. All of the above
compounds are well known to those of skill and are readily
available from commercial sources, by synthesis, or by purification
from natural sources.
In certain embodiments, the vinca alkaloid used in the present
invention is vincristine. Vincristine, also known as leurocristine
sulfate, 22-oxovincaleukoblastine, Kyocristine, vincosid, vincrex,
oncovin, Vincasar PFS.RTM., or VCR, is commercially available from
any of a number of sources, e.g., Pharmacia & Upjohn, Lilly,
IGT, etc. It is often supplied as vincristine sulfate, e.g., as a 1
mg/mL solution.
In other preferred embodiments, the vinca alkaloid used in the
present invention is vinorelbine. Vinorelbine includes vinorelbine
tartrate. Vinorelbine (5'-noranhydrovinblastine) is a semisynthetic
vinca alkaloid structurally distinguished from other members of its
class by the modification of the catharanthine nucleus rather than
the vindoline ring. Vinorelbine has shown efficacy in NSCLC
treatment, alone or in combination with other drugs. Vinorelbine
tartrate (Navelbine.RTM.) is commercially available from Glaxo
Wellcome Inc. (Research Triangle Park, N.C.).
In other preferred embodiments, the vinca alkaloid is vinblastine.
Vinblastine is mainly useful for treating Hodgkin's disease,
lymphocytic lymphoma, histiocytic lymphoma, advanced testicular
cancer, advanced breast cancer, Kaposi's sarcoma, and Letterer-Siwe
disease. Vinblastine is given intravenously to treat Kaposi's
sarcoma, often in combination with other drugs. Vinblastine
(Velban.RTM., Velsar.RTM.) is commercially available from Eli Lilly
(Indianapolis, Ind.).
In another embodiment, liposomal compositions of the present
invention include a taxoid. A taxoid is understood to mean those
compounds that include paclitaxels and docetaxel, and other
chemicals that have the taxane skeleton (Cortes and Pazdur, 1995).
Taxoids may be isolated from natural sources such as the Yew tree
or from cultured cells, or taxoids may be chemically synthesized
molecules. In one embodiment, a taxoid is a chemical of the general
chemical formula, C.sub.47H.sub.51NO.sub.14, including
[2aR-[2a.alpha.,4.beta.,4.alpha.,.beta.,6.beta.,9.alpha.(.alpha-
.R*,.beta.S*),11.alpha.,
12.alpha.,12a.alpha.,12b.alpha.,]]-.beta.-(Benzoylamino)-.alpha.-hydroxyb-
enzenepropanoic acid 6, 12b,
bis(acetyloxy)-12-(benzoyloxy)-2a,3,4,4a,5,6,9,10,11,12,12a,12b-dodeca
hydro-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo-7,11-methano-1H-cyclode-
ca [3,4]benz-[1,2-b]oxet-9-yl ester. More recently, a variety of
water-soluble taxane prodrugs and salts thereof have been
developed, as described, for example, in U.S. Pat. No. 5,981,564,
U.S. Patent Publication No. 20020041897, U.S. Pat. No. 6,380,405,
and PCT Publication No. WO 02/072010. Other examples of taxane
compounds and methods for their preparation are set forth in U.S.
Pat. No. 4,942,184. Additional paclitaxel derivatives contemplated
for use in the present invention include, for example, the water
soluble amino derivatives, including protax-1, described in Mathew
et al., Journal of Medicinal Chemistry 35, 145-151 (1992) and the
taxol prodrugs described in Deutsch et al., Journal of Medicinal
Chemistry, 32 788-792 (1989), including
paclitaxel-C2'-glutaryl-tetramethylene diamine. A variety of other
hydrophilic taxane derivatives, including paclitaxel derivatives,
have been developed, and the invention contemplates the use of any
of these derivatives. Examples of such paclitaxel derivatives
include 2'-O-11-amino-3,6,9,12 tetraoxatetradecanoyl paclitaxel,
2'-O-8-amino-3,6-dioxaoctanoyl paclitaxel, and 2'-O-4-aminohexanoyl
paclitaxelpaclitaxel-C2'-glutaryl-hexamethylene diamine (taxamine).
Further examples include paclitaxel-C2'-glutaryl-di-(glucosamine),
and paclitaxel-C2'C7-di-(glutaryl-di-glucosamine).
In one embodiment, the invention includes liposomal compositions
comprising a camptothecin. Camptothecin (CPT) compounds include
various 20(S)-camptothecins, analogs of 20(S)camptothecin, and
derivatives of 20(S)-camptothecin. Camptothecin, when used in the
context of this invention, includes the plant alkaloid
20(S)-camptothecin, both substituted and unsubstituted
camptothecins, and analogs thereof. Examples of camptothecin
derivatives include, but are not limited to,
9-nitro-20(S)-camptothecin, 9-amino-20(S)-camptothecin,
9-methyl-camptothecin, 9-chlorocamptothecin, 9-flouro-camptothecin,
7-ethyl camptothecin, 10-methylcamptothecin,
10-chloro-camptothecin, 10-bromo-camptothecin,
10-fluoro-camptothecin, 9-methoxy-camptothecin,
11-fluoro-camptothecin, 7-ethyl-10-hydroxy camptothecin,
10,11-methylenedioxy camptothecin, and 10,11-ethylenedioxy
camptothecin, and
7-(4-methylpiperazinomethylene)-10,11-methylenedioxy camptothecin.
Prodrugs of camptothecin include, but are not limited to,
esterified camptothecin derivatives as described in U.S. Pat. No.
5,731,316, such as camptothecin 20-O-propionate, camptothecin
20-O-butyrate, camptothecin 20-O-valerate, camptothecin
20-O-heptanoate, camptothecin 20-O-nonanoate, camptothecin
20-O-crotonate, camptothecin 20-O-2',3'-epoxy-butyrate,
nitrocamptothecin 20-O-acetate, nitrocamptothecin 20-O-propionate,
and nitrocamptothecin 20-O-butyrate. Particular examples of
20(S)-camptothecins include 9-nitrocamptothecin,
9-aminocamptothecin, 10,11-methylendioxy-20(S)camptothecin,
topotecan, irinotecan, SN-38, 7-ethyl-10-hydroxy camptothecin, or
another substituted camptothecin that is substituted at least one
of the 7, 9, 10, 11, or 12 positions.
Camptothecins may optionally be substituted. Substitutions may be
made to the camptothecin scaffold, while still retaining activity.
In certain embodiments, the camptothecin scaffold is substituted at
the 7, 9, 10, 11, and/or 12 positions. Such substitutions may serve
to provide differential activities over the unsubstituted
camptothecin compound. Examples of substituted camptothecins
include 9-nitrocamptothecin, 9-aminocamptothecin,
10,11-methylendioxy20(S)-camptothecin, topotecan, irinotecan,
7-ethyl-10-hydroxy camptothecin, or another substituted
camptothecin that is substituted at least one of the 7, 9, 10, 11,
or 12 positions.
Native, unsubstituted, camptothecin can be obtained by purification
of the natural extract, or may be obtained from the Stehlin
Foundation for Cancer Research (Houston, Tex.). Substituted
camptothecins can be obtained using methods known in the
literature, or can be obtained from commercial suppliers. For
example, 9-nitrocamptothecin may be obtained from SuperGen, Inc.
(San Ramon, Calif.), and 9-aminocamptothecin may be obtained from
Idec Pharmaceuticals (San Diego, Calif.). Camptothecin and various
analogs may also be obtained from standard fine chemical supply
houses, such as Sigma Chemicals.
In an additional embodiment, the invention includes liposomal
compositions comprising etoposide. Etoposide (also referred to as
VP-16, VP-16-213, or VePesid.RTM.), a semi-synthetic
podophyllotoxin derived from the root of Podophyllum peltatum
(mandrake plant), is a widely used cancer chemotherapy drug that is
approved for clinical use in non-Hodgkin's lymphoma, small cell
lung cancer, and refractory testicular cancer. In another
embodiment, the liposomal composition of the invention comprises
the etoposide derivative, NK611, or a pharmaceutically acceptable
salt thereof.
While liposomal compositions of the invention generally comprise a
single active agent, in certain embodiments, they may comprise more
than one active agent.
2. Methods of Loading Liposomes
Liposomal formulations of the invention are generally prepared by
loading an active agent into liposomes. Loading may be accomplished
by any means available in the art, including those described in
further detail below. Furthermore, the invention contemplates the
use of either passive or active loading methods.
Passive loading generally requires addition of the drug to the
buffer at the time the liposomes are formed or reconstituted. This
allows the drug to be trapped within the liposome interior, where
it will remain if it is not lipid soluble and if the vesicle
remains intact (such methods are described, e.g., in PCT
Publication No. WO 95/08986). The buffer which is used in the
formation of the liposomes can be any biologically compatible
buffer solution of, for example, isotonic saline, phosphate
buffered saline, dextrose solutions (e.g., 5% dextrose) or other
low ionic strength buffers. The resulting liposomes encompassing
the active agent can then be sized as described above.
In the case of hydrophobic drugs, the drug and liposome components
can be dissolved in an organic solvent in which all species are
miscible and concentrated to a dry film. A buffer is then added to
the dried film and liposomes are formed having the drug
incorporated into the lipid bilayer. The liposomes containing the
bilayer-inserted drug can then be sized as described above.
Liposomal compositions of the invention may also be prepared using
active loading methods. Numerous methods of active loading are
known to those of skill in the art. Such methods typically involve
the establishment of some form of gradient that draws lipophilic
compounds into the interior of liposomes where they can reside for
as long as the gradient is maintained. Very high quantities of the
desired active agent can be obtained in the interior. At times, the
active agent may precipitate out in the interior. A wide variety of
active agents can be loaded into liposomes with encapsulation
efficiencies approaching 100% by using active loading methods
involving a transmembrane pH or ion gradient (see, Mayer, et al.,
Biochim. Biophys. Acta 1025:143-151 (1990) and Madden, et al.,
Chem. Phys. Lipids 53:37-46 (1990)).
Transmembrane potential loading has been described in detail in
U.S. Pat. Nos. 4,885,172; 5,059,421; 5,171,578; and 5,837,282
(which teaches ionophore loading). Briefly, the transmembrane
potential loading method can be used with essentially any active
agent, including, e.g., conventional drugs, that can exist in a
charged state when dissolved in an appropriate aqueous medium. In
certain embodiments, the active agent will be relatively lipophilic
facilitating diffusion across the lipid bilayer. A transmembrane
potential is created across the bilayers of the liposomes or
protein-liposome complexes and the active agent is loaded into the
liposome by means of the transmembrane potential. The transmembrane
potential is generated by creating a concentration gradient for one
or more charged species (e.g., Na.sup.+, K.sup.+, and/or H.sup.+)
across the membranes. This concentration gradient is generated by
producing liposomes having different internal and external media
and has an associated proton gradient. Active agent accumulation
can then occur in a manner predicted by the Henderson-Hasselbach
equation.
One particular method of loading active agents, including, e.g.,
vinca alkaloids, to produce a liposomal composition of the present
invention is ionophore-mediated loading, as disclosed and claimed
in U.S. Pat. No. 5,837,282. One example of an ionophore used in
this procedure is A23187. Liposomes can be formed including a
divalent cation, such as magnesium or manganese, in the aqueous
interior. External divalent cation is then removed creating a
chemical gradient across the liposomal bilayer. Addition of A23187
to the liposomes facilitates transfer of divalent cation out of the
liposomes and hydrogen ion transport into the liposomes in a 1:2
ratio (i.e., no net charge transfer). As ionophore-mediated loading
is an electroneutral process, there is no transmembrane potential
generated.
Accordingly, the invention provides methods of loading liposomes
via ionophore-mediated loading. Similarly, the invention provides
methods of preparing or manufacturing a liposomal composition of
the invention comprising loading a liposome comprising DHSM with a
therapeutic agent according to the method of loading liposomes
described here, including ionophore-mediated loading. The invention
also provides any of the liposomes described herein comprising a
divalent cation, such as Mn.sup.2+ or Mg.sup.2+ in their
interior.
While ionophore-mediated loading methods have been generally
described in U.S. Pat. No. 5,837,282, it was surprisingly
discovered that increased drug encapsulation is achieved by using
certain specific conditions within the general ranges previously
disclosed. As shown in Table 2, increased encapsulation is achieved
using either the divalent metal ion, Mn.sup.2+, increased salt
concentration, higher loading temperatures, or a combination of
such conditions.
TABLE-US-00002 TABLE 2 Percent NK611 encapsulation via
ionophore-mediated loading SM/Chol DHSM/Chol D/L ratio Buffer Temp
(.degree. C.) (% encap.) (% encap.) 0.4 300 mM MgSO.sub.4 60 78 14
0.4 300 mM MnSO.sub.4 60 88-93 74-83 0.4 300 mM MnSO.sub.4 70
n.d..sup.1 82-93 1.0 300 mM MgSO.sub.4 60 45 n.d. 1.0 300 mM
MnSO.sub.4 60 53 n.d. 1.0 600 mM MnSO.sub.4 70 n.d..sup. 79-90
.sup.1n.d. indicates not determined
Thus, the present invention includes methods of loading an active
agent (e.g., a therapeutic agent) into a liposome, wherein the
liposome has an encapsulated medium comprising MnSO.sub.4, by
incubating said liposomes with an external solution comprising the
active agent and an ionophore. Thus, in one preferred embodiment,
the divalent metal ion is Mn.sup.2+, and in certain preferred
embodiments, the MnSO.sub.4 is present at a concentration equal or
greater than 300 mM or in the range from 300 mM to 600 mM. In
particular preferred embodiments, the MnSO.sub.4 is present at a
concentration of either 300 mM or 600 mM.
In additional embodiments, the loading is performed at a
temperature of at least 60.degree. C., at least 65.degree. C., or
at least 70.degree. C. In particular embodiments, loading is
performed at a temperature in the range of 60.degree. to
70.degree., and in certain embodiments, loading is performed at
either 60.degree. C. or 70.degree. C. Loading may be performed in
the presence of any concentration of active agent (e.g., drug), or
at any desired drug to lipid ratio, including any of the drug to
lipid ratios described herein. In certain embodiments, loading is
performed at a drug to lipid ratio within the range of 0.005
drug:lipid (by weight) to about 1.0 drug:lipid (by weight). In
particular embodiments, loading is performed at a drug to lipid
ratio within the range of 0.075 drug:lipid (by weight) to 0.20
drug:lipid (by weight). In other particular embodiments, loading is
performed at a drug to lipid ratio of between 0.2 drug:lipid (by
weight) to 0.4 drug:lipid (by weight). In other particular
embodiments, loading is performed at between 0.4 drug:lipid (by
weight) and 1.0 drug:lipid (by weight).
In additional specific embodiments, the preferred loading methods
are used to load liposomes of the invention, i.e., liposomes
comprising DHSM, wherein at least 50% of the total phospholipids
present in the liposomes are DHSM. Thus, in one particular
embodiment, the invention includes a method of ionophore-mediated
loading of a therapeutic agent into a liposome comprising DHSM,
wherein said DHSM comprises at least 50% of the total phospholipids
of the liposome and wherein said liposomes have an encapsulated
medium comprising 300 mM MnSO.sub.4, comprising incubating said
liposomes with an external solution comprising said therapeutic
agent and an ionophore at a temperature of 70.degree. C. to form
therapeutic agent-loaded liposomes.
The present invention also provides methods of preparing liposomal
compositions and methods of making or manufacturing liposomal
compositions of the present invention. In general, such methods
comprise loading a liposome of the present invention with an active
agent. Loading may be accomplished by any means available in the
art, including those described herein, and, particularly,
ionophore-mediated loading methods described here. Such methods may
further comprise formulating the resulting composition to produce a
pharmaceutical composition suitable for administration to a
subject.
In one embodiment, a method of the invention comprises loading a
liposome comprising DHSM with a therapeutic agent. In a related
embodiment, a method of the invention comprises producing a
liposome comprising DHSM or and loading the liposome with a
therapeutic agent. In specific embodiments, the liposomes have
additional components or characteristics as described in the
instant application.
3. Characteristics of Liposomal Compositions
Liposomal compositions of the present invention may be
characterized in a variety of ways, based, in part, upon their
lipid and active agent components.
One important characteristic of liposomal compositions used for
pharmaceutical purposes is the drug:lipid ratio. The rate of drug
release from the liposomes may be decreased by increasing the
drug:lipid ratio and thereby causing precipitation of a proportion
of the encapsulated drug (see U.S. Patent Publication No.
2002/0119990-A1). As the drug:lipid ratio is increased however,
lower lipid doses are administered to a patient to achieve the
desired drug dose. This may result in faster clearance of the
drug-loaded liposomes from the plasma and hence reduce drug
delivery to disease sites, including tumor sites. Addition of empty
liposomes (liposomes containing no drug) to drug-loaded liposomes
can allow administration of a suitable lipid dose to prevent rapid
clearance from the plasma while maintaining slow drug release from
the drug-loaded liposomes (see U.S. Patent Publication No.
2002/0119990-A1). Techniques for generating specific drug:lipid
ratios are well known in the art. The drug:lipid ratio can be
varied by using appropriate concentrations of drug and liposomes
during the drug loading procedure, as described, for example, in
Mayer et al., Cancer Res. 49: 5922-5930 (1989).
In the present invention, it is envisaged that for different
applications, different drug:lipid ratios may be desired.
Drug:lipid ratios can be measured on a weight to weight basis, a
mole to mole basis or any other designated basis.
In certain embodiments, drug:lipid ratios range from 0.005
drug:lipid (by weight) to about 1.0 drug:lipid (by weight). In
other embodiments, drug:lipid ratios range from 0.075 drug:lipid
(by weight) to 0.20 drug:lipid (by weight). In other particular
embodiments, drug:lipid ratios range from between 0.2 drug:lipid
(by weight) to 0.4 drug:lipid (by weight). In other particular
embodiments, drug:lipid ratios range between 0.4 drug:lipid (by
weight) and 1.0 drug:lipid (by weight). In particular embodiments,
the drug:lipid ratio is approximately 0.05 (by weight) when the
drug is vincristine, approximately 0.3 (by weight) when the drug is
vinorelbine, approximately 0.1 (by weight) when the drug is
topotecan, approximately 0.4 (by weight) when the drug is NK611,
and approximately 0.1 (by weight) when the drug is taxol.
In certain embodiments, liposomal compositions of the present
invention comprise both empty liposomes and liposomes loaded with
one or more active agents. In a particular embodiment, a liposomal
composition of the present invention comprises empty liposomes, and
liposomes loaded with one or more active agents, wherein the loaded
liposomes comprise DHSM and an internal buffer comprising
MnSO.sub.4 or Mn.sup.2+.
Liposomal compositions used for pharmaceutical purposes are often
intended to modify drug biodistribution, drug half-life in plasma,
drug stability in plasma, or duration of drug exposure to target
cells (e.g., tumor cells). Ultimately such changes in drug
pharmacokinetics are expected to result in increased drug activity,
for example, increased antitumor activity. Characterization of the
liposomal compositions of the present invention therefore
appropriately includes pharmacokinetics evaluation, determination
of drug release from the liposomes in vitro and/or in vivo, and
determination of the therapeutic activity of the liposomal drug.
Accordingly, in various embodiments, liposomes and liposomal
compositions of the present invention retain at least 10%, at least
15%, at least 20%, at least 30%, at least 40%, at least 50%, at
least 60%, at least 70%, at least 80%, or at least 90% of active
compound at one hour, as determined by the in vitro release (IVR)
method described in Example 5. In other embodiments, liposomes and
liposomal compositions of the present invention retain at least
20%, at least 30%, at least 40%, at least 50%, at least 60%, at
least 70%, at least 80%, or at least 90% of active compound at
four, eight or twenty-four hours, as determined by the IVR method
described in Example 5. Similarly, in certain embodiments,
liposomes and liposomal compositions of the present invention are
associated with active agent plasma retention of at least 10%, at
least 15%, at least 20%, at least 30%, at least 40%, at least 50%,
at least 60%, at least 70%, at least 80%, or at least 90% four
hours post injection or at least 10%, at least 15%, at least 20%,
at least 30%, at least 40%, at least 50%, at least 60%, at least
70%, at least 80%, or at least 90% eight or twenty-four hours post
injection, as measured in an in vivo model, such as that described
in Examples 6-8.
In other embodiments of the invention, the liposomes or liposomal
compositions of the invention have a plasma circulation half-life
of at least 0.5, 0.8, 1.2, 1.5, 2.0, 4.0, 6.0, 8.0, or 12 hours. In
other embodiments, liposomal compositions of the present invention
have a plasma drug half-life of at least 0.5, 0.8, 1.2, 1.5, 2.0,
4.0, 6.0, 8.0, or 12 hours. Circulation and blood or plasma
clearance half-lives may be determined as described, for example,
in U.S. Patent Publication No. 2004-0071768-A1.
In related embodiments, the circulation half-life of encapsulated
vinorelbine in blood is at least 0.8 hours, or the time required
for 50% release of encapsulated vinorelbine from the liposomes in
blood is at least 2.0 hours.
In related embodiments, the circulation half-life of encapsulated
topotecan in blood is at least 1.0 hour, or the time required for
50% release for encapsulated topotecan from the liposomes in blood
is at least 2.0 hours.
As described herein, it was a surprising finding of the present
invention that liposomal compositions comprising DHSM or Mn.sup.2+
as the internal cation exhibit increased drug retention as compared
to liposomal compositions comprising ESM or Mg.sup.2+. In certain
embodiments, therefore, the present invention provides liposomal
compositions comprising an active agent and MnSO.sub.4 or Mn.sup.2+
in the interior of the liposomes. In a related embodiment, the salt
or divalent cation in the interior of liposomes comprising an
active agent is MnSO.sub.4 or Mn.sup.2+. In one embodiment, the
active agent is topotecan, and the present invention includes a
liposomal composition comprising topotecan and MnSO.sub.4 or
Mn.sup.2+ in the interior of the liposomes. Furthermore, it was a
surprising finding of the present invention that Mn.sup.2+ and DHSM
exhibited an additive effective in increasing drug retention.
Accordingly, in one particular embodiment, liposomal compositions
of the present invention comprise both DHSM and Mn.sup.2+. Such
liposomes may further comprise a therapeutic agent, such as, e.g.,
topotecan. Liposomal compositions comprising MnSO.sub.4 or
Mn.sup.2+ can be prepared essentially as known in the art, and as
described herein, e.g., by substituting MnSO.sub.4 for other salts,
such as MgSO.sub.4.
The present invention also provides liposomes and liposomal
compositions in kit form. The kit may comprise a ready-made
formulation or a formulation that requires mixing before
administration. The kit will typically comprise a container that is
compartmentalized for holding the various elements of the kit. The
kit will contain the liposomes or liposomal compositions of the
present invention or the components thereof, in hydrated or
dehydrated form, with instructions for their rehydration and
administration. In particular embodiments, a kit comprises at least
one compartment containing a liposome of the present invention that
is loaded with an active agent. In another embodiment, a kit
comprises at least two compartments, one containing a liposome of
the invention and the other containing an active agent. Of course,
it is understood that any of these kits may comprise additional
compartments, e.g., a compartment comprising a buffer, such as
those described in U.S. Patent Publication No. 2004-0228909-A1.
Kits of the present invention, which comprise liposomes comprising
DHSM, may also contain other features of the kits described in U.S.
Patent Publication No. 2004-0228909 A1. Further the kit may contain
drug-loaded liposomes in one compartment and empty liposomes in a
second compartment. Alternatively, the kit may contain a liposome
of the present invention, an active agent to be loaded into the
liposome of the present invention in a second compartment, and an
empty liposome in a third compartment.
In a particular embodiment, a kit of the present invention
comprises a therapeutic compound encapsulated in a liposome
comprising DHSM, wherein said DHSM constitutes at least 20% or at
least 50% (molar basis) of total phospholipids present in the
liposome, as well as an empty liposome. In one embodiment, the
liposome containing therapeutic compound and the empty liposome are
present in different compartments of the kit.
C. Liposomal Delivery of Active Agents
The liposomal compositions described above may be used for a
variety of purposes, including the delivery of an active agent or
therapeutic agent or compound to a subject or patient in need
thereof. Subjects include both humans and non-human animals. In
certain embodiments, subjects are mammals. In other embodiments,
subjects are one or more particular species or breed, including,
e.g., humans, mice, rats, dogs, cats, cows, pigs, sheep, or
birds.
Thus, the present invention also provides methods of treatment for
a variety of diseases and disorders, as well as methods related to
cosmetic purposes, including, but not limited to, methods of
applying cosmetics and methods of providing cosmetics, makeup
products, moisturizers or other compounds, including, e.g., those
intended to provide a cosmetic benefit.
1. Methods of Treatment
The liposomal compositions of the present invention may be used to
treat any of a wide variety of diseases or disorders, including,
but not limited to, inflammatory diseases, cardiovascular diseases,
nervous system diseases, tumors, demyelinating diseases, digestive
system diseases, endocrine system diseases, reproductive system
diseases, hemic and lymphatic diseases, immunological diseases,
mental disorders, muscoloskeletal diseases, neurological diseases,
neuromuscular diseases, metabolic diseases, sexually transmitted
diseases, skin and connective tissue diseases, urological diseases,
and infections.
In one embodiment, the liposomal compositions and methods described
herein can be used to treat any type of cancer. In particular,
these methods can be applied to cancers of the blood and lymphatic
systems, including lymphomas, leukemia, and myelomas. Examples of
specific cancers that may be treated according to the invention
include, but are not limited to, Hodgkin's and non-Hodgkin's
Lymphoma (NHL), including any type of NHL as defined according to
any of the various classification systems such as the Working
formulation, the Rappaport classification and, preferably, the REAL
classification. Such lymphomas include, but are not limited to,
low-grade, intermediate-grade, and high-grade lymphomas, as well as
both B-cell and T-cell lymphomas. Included in these categories are
the various types of small cell, large cell, cleaved cell,
lymphocytic, follicular, diffuse, Burkitt's, Mantle cell, NK cell,
CNS, AIDS-related, lymphoblastic, adult lymphoblastic, indolent,
aggressive, transformed and other types of lymphomas. The methods
of the present invention can be used for adult or childhood forms
of lymphoma, as well as lymphomas at any stage, e.g., stage I, II,
III, or IV. The various types of lymphomas are well known to those
of skill, and are described, e.g., by the American Cancer Society
(see, e.g., www3.cancer.org).
The compositions and methods described herein may also be applied
to any form of leukemia, including adult and childhood forms of the
disease. For example, any acute, chronic, myelogenous, and
lymphocytic form of the disease can be treated using the methods of
the present invention. In preferred embodiments, the methods are
used to treat Acute Lymphocytic Leukemia (ALL). More information
about the various types of leukemia can be found, inter alia, from
the Leukemia Society of America (see, e.g., www.leukemia.org).
Additional types of tumors can also be treated using the methods
described herein, such as neuroblastomas, myelomas, prostate
cancers, small cell lung cancer, colon cancer, ovarian cancer,
non-small cell lung cancer, brain tumors, breast cancer, and
others.
The liposomal compositions of the invention may be administered as
first line treatments or as secondary treatments. In addition, they
may be administered as a primary chemotherapeutic treatment or as
adjuvant or neoadjuvant chemotherapy. For example, treatments of
relapsed, indolent, transformed, and aggressive forms of
non-Hodgkin's Lymphoma may be administered following at least one
course of a primary anti-cancer treatment, such as chemotherapy
and/or radiation therapy.
2. Administration of Liposomal Compositions
Liposomal compositions of the invention are administered in any of
a number of ways, including parenteral, intravenous, systemic,
local, oral, intratumoral, intramuscular, subcutaneous,
intraperitoneal, inhalation, or any such method of delivery. In one
embodiment, the compositions are administered parenterally, i.e.,
intraarticularly, intravenously, intraperitoneally, subcutaneously,
or intramuscularly. In a specific embodiment, the liposomal
compositions are administered by intravenous infusion or
intraperitoneally by a bolus injection. For example, in one
embodiment, a patient is given an intravenous infusion of the
liposome-encapsulated active agent through a running intravenous
line over, e.g., 5-10 minutes, 15-20 minutes, 30 minutes, 60
minutes, 90 minutes, or longer. In one embodiment, a 60 minute
infusion is used. In other embodiments, an infusion ranging from
6-10 or 15-20 minutes is used. Such infusions can be given
periodically, e.g., once every 1, 3, 5, 7, 10, 14, 21, or 28 days
or longer, preferably once every 7-21 days, and preferably once
every 7 or 14 days.
Liposomal compositions of the invention may be formulated as
pharmaceutical compositions suitable for delivery to a subject. The
pharmaceutical compositions of the invention will often further
comprise one or more buffers (e.g., neutral buffered saline or
phosphate buffered saline), carbohydrates (e.g., glucose, mannose,
sucrose, dextrose or dextrans), mannitol, proteins, polypeptides or
amino acids such as glycine, antioxidants, bacteriostats, chelating
agents such as EDTA or glutathione, adjuvants (e.g., aluminum
hydroxide), solutes that render the formulation isotonic, hypotonic
or weakly hypertonic with the blood of a recipient, suspending
agents, thickening agents and/or preservatives. Alternatively,
compositions of the present invention may be formulated as a
lyophilizate.
The concentration of drug and liposomes in the pharmaceutical
formulations can vary widely, i.e., from less than about 0.05%,
usually at or at least about 2-5% to as much as 10 to 30% by weight
and will be selected depend upon the particular drug used, the
disease state being treated and the judgment of the clinician
taking. Further, the concentration of drug and liposomes will also
take into consideration the fluid volume administered, the
osmolality of the administered solution, and the tolerability of
the drug and liposomes. In some instances it may be preferable to
use a lower drug or liposome concentration to reduce the incidence
or severity of infusion-related side effects.
Suitable formulations for use in the present invention can be
found, e.g., in Remington's Pharmaceutical Sciences, Mack
Publishing Company, Philadelphia, Pa., 17.sup.th Ed. (1985). Often,
intravenous compositions will comprise a solution of the liposomes
suspended in an acceptable carrier, such as an aqueous carrier. Any
of a variety of aqueous carriers can be used, e.g., water, buffered
water, 0.4% saline, 0.9% isotonic saline, 0.3% glycine, 5%
dextrose, and the like, and may include glycoproteins for enhanced
stability, such as albumin, lipoprotein, globulin, etc. Often,
normal buffered saline (135-150 mM NaCl) or 5% dextrose will be
used. These compositions can be sterilized by conventional
sterilization techniques, such as filtration. The resulting aqueous
solutions may be packaged for use or filtered under aseptic
conditions and lyophilized, the lyophilized preparation being
combined with a sterile aqueous solution prior to administration.
The compositions may also contain pharmaceutically acceptable
auxiliary substances as required to approximate physiological
conditions, such as pH adjusting and buffering agents, tonicity
adjusting agents and the like, for example, sodium acetate, sodium
lactate, sodium chloride, potassium chloride, calcium chloride,
etc. Additionally, the composition may include lipid-protective
agents, which protect lipids against free-radical and
lipid-peroxidative damages on storage. Lipophilic free-radical
quenchers, such as .alpha.-tocopherol and water-soluble
iron-specific chelators, such as ferrioxamine, are suitable.
The amount of active agent administered per dose is selected to be
above the minimal therapeutic dose but below a toxic dose. The
choice of amount per dose will depend on a number of factors, such
as the medical history of the patient, the use of other therapies,
and the nature of the disease. In addition, the amount of active
agent administered may be adjusted throughout treatment, depending
on the patient's response to treatment and the presence or severity
of any treatment-associated side effects. In certain embodiments,
the dosage of liposomal composition or the frequency of
administration is approximately the same as the dosage and schedule
of treatment with the corresponding free active agent. However, it
is understood that the dosage may be higher or more frequently
administered as compared to free drug treatment, particularly where
the liposomal composition exhibits reduced toxicity. It is also
understood that the dosage may be lower or less frequently
administered as compared to free drug treatment, particularly where
the liposomal composition exhibits increased efficacy as compared
to the free drug. Exemplary dosages and treatment for a variety of
chemotherapy compounds (free drug) are known and available to those
skilled in the art and are described in, e.g., Physician's Cancer
Chemotherapy Drug Manual, E. Chu and V. Devita (Jones and Bartlett,
2002).
Patients typically will receive at least two courses of such
treatment, and potentially more, depending on the response of the
patient to the treatment. In single agent regimens, total courses
of treatment are determined by the patient and physician based on
observed responses and toxicity.
3. Combination Therapies
In numerous embodiments, liposomal compositions of the invention
will be administered in combination with one or more additional
compounds or therapies, such as surgery, radiation treatment,
chemotherapy, or other active agents, including any of those
described above. Liposomal compositions may be administered in
combination with a second active agent for a variety of reasons,
including increased efficacy or to reduce undesirable side effects.
The liposomal composition may be administered prior to, subsequent
to, or simultaneously with the additional treatment. Furthermore,
where a liposomal composition of the present invention (which
comprises a first active agent) is administered in combination with
a second active agent, the second active agent may be administered
as a free drug, as an independent liposomal formulation, or as a
component of the liposomal composition comprising the first drug.
In certain embodiments, multiple active agents are loaded into the
same liposomes. In other embodiments, liposomes comprising an
active agent are used in combination with one or more free drugs.
In particular embodiments, liposomal compositions comprising an
active agent are formed individually and subsequently combined with
other compounds for a single co-administration. Alternatively,
certain therapies are administered sequentially in a predetermined
order, such as in CHOP or lipo-CHOP, described further below.
Accordingly, liposomal compositions of the present invention may
comprise one or more active agents.
In one embodiment of combination treatment according to the present
invention, multiple vinca alkaloids are co-administered, or one or
more vinca alkaloids is administered in conjunction with another
therapeutic compound, such as cyclophosphamide, dexamethasone,
doxorubicin, prednisone, other antineoplastics such as the taxanes,
camptothecins, and/or podophyllins, other chemotherapeutic agents
such as antisense drugs or anti-tumor vaccines. In one embodiment,
liposome encapsulated vincristine is used along with
cyclophosphamide, doxorubicin, and prednisone, thereby forming an
improved CHOP formulation comprising liposome encapsulated
vincristine ("lipo-CHOP"). In a related embodiment, lipo-CHOP is
used in combination with one or more additional therapeutic
compounds, such as Rituxan.TM. (IDEC Pharmaceuticals). In another
embodiment, liposome encapsulated vincristine is used in
combination with prednisone.
In other embodiments, liposomal vinorelbine is used in combination
with one or more other chemotherapeutic agents, such as Gemcitabine
or taxol or derivatives thereof. Combination therapies including
vinorelbine have been demonstrated to have increased efficacy as
compared to single drug treatment, in certain cases. For example,
vinorelbine is associated with promising six-month and median
survival rates in women with ovarian cancer that has relapsed
following treatment with a platinum and paclitaxel, and the
combination of vinorelbine and cisplatin has shown superior results
in terms of response rates and survival when compared to
single-agent cisplatin. Wozniak, A. J. et al., J. Clin. Oncol.
16:2459-2465 (1998).
Liposomal compositions of the invention, including, e.g.,
liposome-encapsulated vinca alkaloids, can also be combined with
anti-tumor agents such as monoclonal antibodies including, but not
limited to, Oncolym.TM. (Techniclone Corp. Tustin, Calif.) or
Rituxan.TM. (IDEC Pharmaceuticals), Bexxar.TM. (Coulter
Pharmaceuticals, Palo Alto, Calif.), IDEC-Y2B8 (IDEC
Pharmaceuticals Corporation), Erbitux.TM. (Imclone Systems Inc.)
and Avastin.TM. (Genentech Corp.).
In a preferred embodiment, liposomal compositions of the present
invention are administered in combination with an anti-cancer
compound or therapy that provides an increased or synergistic
improvement in tumor reduction based on mechanism of action and
non-overlapping toxicity profiles. For example, liposomal vinca
alkaloids can be delivered with a taxane, which optionally may also
be a liposomal taxane. While it is thought that vinca alkaloids
depolymerize microtubules and taxanes stabilize microtubules, the
two compounds have been found to act synergistically in the
impairment of tumor growth, presumably because both are involved in
the inhibition of microtubule dynamics. See, Dumontet, C. and
Sikic, B. I. (1999) J. Clin Onc. 17(3) 1061-1070. Liposomal
formulations of the vinca alkaloids according to the present
invention will thus significantly diminish the myeloid and
neurologic toxicity associated with the sequential administration
of free form vinca alkaloids and taxanes.
Other combination therapies known to those of skill in the art can
be used in conjunction with the methods of the present
invention.
Examples of drugs used in combination with conjugates and other
chemotherapeutic agents to combat undesirable side effects of
cancer or chemotherapy include zoledronic acid (Zometa) for
prevention of bone metastasis and treatment of high calcium levels,
Peg-Filgrastim for treatment of low white blood count, SDZ PSC 833
to inhibit multidrug resistance, and NESP for treatment of
anemia.
EXAMPLE 1
Preparation of Liposomes Comprising Dihydrosphingomyelin
Dihydrosphingomyelin can be prepared by hydrogenation of
sphingomyelin. By way of example, details are provided below for
preparation of egg dihydrosphingomyelin (EDHSM) and
D-erythro-N-palmitoyl-dihydrosphingomyelin (16:0-DHSM).
Egg Dihydrosphingomyelin.
Essentially, egg sphingomyelin (ESM) (25 g) was dissolved in
ethanol (250 mL) in a round-bottomed flask. 10% palladium/carbon
catalyst (2.5 g) was added, and the flask was sealed with a rubber
septum. The flask was flushed with argon for 30 minutes. Hydrogen
was slowly passed through the system, using a bubbler to prevent
flowback of air into the reaction mixture. The reaction was warmed
in a water bath at approximately 40.degree. C. on a stirrer for
approximately two hours and then flushed with argon to remove
excess hydrogen. Cyclohexene (5 mL) was added to quench any active
catalyst remaining. The suspension was filtered through
diatomaceous earth, observing the proper precautions when filtering
pyrophoric solids. The filtrate was dried down on a rotovap, and
the residue dissolved in warmed ethanol (100 mL). The solution was
cooled and acetone (100 mL) added. The solution was cooled to room
temperature, and the resultant precipitate filtered off under
vacuum. The precipitation was repeated, and the resultant product
dried under vacuum, yielding 14 g of purified dihydrosphingomyelin
(DHSM).
DHSM prepared according to the above procedure was analyzed by
nuclear magnetic resonance (NMR) and high pressure liquid
chromatography (HPLC). NMR spectra analysis of two batches of DHSM
prepared from egg sphingomyelin indicated that less than 1% of the
double bonds present in the egg sphingomyelin starting material
were still present in the prepared DHSM. HPLC analysis of two
batches of DHSM prepared from egg sphingomylein and one batch of
DHSM prepared from brain sphingomyelin demonstrated that greater
than 97% of the prepared DHSM possessed a 16:0, 18:0, 20:0, 22:0,
23:0 or 24:0 N-acyl chain. Conversion of sphingosine to
dihydrosphingosine was efficient with only about 0.5-1.0% of the
starting material present in the final product (based on
D-erythro-N-palmityl-sphingomyelin).
D-erythro-N-palmityl-dihydrosphingomyelin (16:0-DHSM)
To prepare 16:0-DHSM, D-erythro-N-palmityl-sphingomyelin (16:0-SM)
was purified from egg yolk sphingomyelin (Avanti Polar Lipids, Inc.
(Alabaster, Ala.) by reverse-phase HPLC (LiChrospher 100 RP-18
columns, 5 .mu.m particle size, 240.times.4 mm column dimensions;
Merck, Darmstadt, Germany) using 5 vol-% water in methanol as
eluent (at 1 ml/min, column temperature 40.degree. C.).
D-erythro-N-palmityl-dihydrosphingomyelin (16:0-DHSM) was prepared
from 16:0-SM by hydrogenation using palladium oxide (Aldrich
Chemical Co., Milwaukee, Wis.), as catalyst (Schneider and Kennedy,
J. Lipid Res. 8:202-209 (1967)), and purified as described for egg
dihydrosphingomyelin.
Liposomes comprising sphingomyelin derived from various sources or
dihydrosphingomyelin (DHSM) were generated according to standard
procedures. Liposomes comprising SM or DHSM were prepared
essentially as previously described in Fenske, D. B. et al.,
Biochim Biophys Acta. 1414(1-2):188-204 (1998) and Webb, M. S. et
al., Br J Cancer 72(4):896-904 (1995), using ethanol as described
in Boman, N. L. et al., Cancer Res. 54(11):2830-3 (1994).
EXAMPLE 2
Differential Scanning Calorimetry (DSC) of Liposomes Comprising
Dihydrosphingomyelin
The thermotropic properties of various dihydrosphingomyelins and
the corresponding sphingomyelins were characterized using
differential scanning calorimetry. Large multilamellar liposomes
composed of the various DHSM and SM species were prepared in
distilled water at a phospholipid concentration of 15 mg/mL. Before
loading in DSC cells, samples were brought to room temperature and
then degassed under vacuum with stirring for 5 minutes. Immediately
prior to loading in DSC cells, samples were vortexed to achieve
homogeneity. Scans were performed on a MC-DSC 4100 calorimeter from
Calorimetry Sciences Corporation, using a heating or cooling rate
of 5.degree. C./hour. Generally both heating and cooling scans were
performed over the temperature range 20-60.degree. C.
Concentration-normalized DSC scans for ESM and EDHSM are shown in
FIG. 1. The scans show a gel to liquid-crystalline phase transition
for both liposomal compositions. The transition temperature (Tc)
for ESM is approximately 34.degree. C., while the Tc for EDHSM is
approximately 46.degree. C. A narrow, single transition is seen for
EDHSM and a distinct pre-transition is seen at about 38.degree.
C.
Concentration-normalized DSC scans for milk sphingomyelin (MSM) and
milk dihydrosphingomyelin (MDHSM) are shown in FIG. 2. The scans
show a gel to liquid-crystalline phase transition for both
liposomal compositions. The phase transition for MSM is broad with
a Tc of approximately 26.degree. C. In the case of MDHSM, two
distinct transitions are seen with the first Tc being about
34.degree. C. The presence of two endotherms suggests that phase
separation may occur in liposomes composed of MDHSM when these are
cooled below the Tc. It is noted that the N-acyl chains in MDHSM
are predominantly C22:0, C23:0 and C24:0 with relatively small
quantities of C16:0 or C18:0 present (Table 1). Without wishing to
be bound by any particular theory, the difference in chain length
between the dihydrosphingosine long chain base and the N-acyl
chains may contribute to the complex thermotropic behavior observed
with MDHSM.
Concentration-normalized DSC scans for brain sphingomyelin (BSM)
and brain dihydrosphingomyelin (BDHSM) are shown in FIG. 3. The
scans show a gel to liquid-crystalline phase transition for both
liposomal compositions. The phase transition for BSM is broad with
a Tc of about 30.degree. C. A narrow, single transition is seen for
BDHSM with a Tc of approximately 48.degree. C. and a distinct
pre-transition is seen at about 35.degree. C.
EXAMPLE 3
Loading of Liposomes Comprising Dihydrosphingomyelin with Topotecan
at Various Temperatures
The loading efficiency at different temperatures of liposomes
prepared using either DHSM or SM was compared as follows.
Topotecan was loaded into liposomes comprising either DHSM or SM
and cholesterol at a 55:45 (mol/mol) ratio using the
A23187-ionophore method.
Lipids (ESM or EDHSM and cholesterol) were dissolved in ethanol at
65.degree. C. at a ESM:Chol or EDHSM:Chol ratio of 55:45 mol ratio.
Multilamellar vesicles (MLV) were formed by adding the hot lipid
solution as a steady stream by injection with a syringe over
.about.30 seconds with mixing to a 353 mM MgSO.sub.4/235 mM sucrose
or 353 mM MnSO.sub.4/235 mM sucrose solution.
The MLV were extruded at 65.degree. C. through two stacked 80 nm
polycarbonate membranes by applying nitrogen gas pressure
(.about.200 psi) to a 10 or 100 ml Extruder. Extrusion was repeated
until a vesicle size of 90 to 110 nm was achieved, which usually
required 4 to 6 passes. Vesicle size was determined by
quasi-elastic light scattering using a Nicomp 380 submicron
particle sizer (Santa Barbara, Calif.).
The resulting large unilamellar vesicle (LUV) formulation was
dialyzed against 300 mM sucrose to remove the residual ethanol and
external magnesium sulphate using a tangential flow ultrafiltration
system (20 wash volumes). The final preparation was concentrated to
50 mg/ml and stored at 5.degree. C. until required for loading.
For topotecan loading, EDTA and phosphate buffer were added to
liposomes (15 mg/ml total lipid, pH 6.5) at 25 mM and 50 mM final
concentrations, respectively. The liposome suspensions were
pre-heated to 60.degree. C. using a water bath before the ionophore
(1 .mu.g A23187/mg lipid) was added. After a 10 min incubation, a
10 mg/ml topotecan stock solution (solubilized in 1 mg/ml tartaric
acid) was added.
At 60.degree. C., ESM/CH (55:45) vesicles containing either
Mg.sup.2+ or Mn.sup.2+ loaded rapidly with >90% uptake occurring
within 15 min. (FIG. 4A). In contrast, topotecan uptake into
EDHSM/CH (55:45) vesicles at 60.degree. C. was significantly slower
(FIG. 4A), requiring a minimum incubation time of .about.40 min. to
achieve .about.90% loading of vesicles containing either Mg.sup.2+
or Mn.sup.2+. Higher overall encapsulation efficiencies were
observed for Mn.sup.2+-containing vesicles.
To determine if the slower uptake kinetics with EDHSM/CH (55:45)
vesicles could be overcome by using a higher loading temperature,
DHSM/CH (55:45) vesicles containing either Mg.sup.2+ or Mn.sup.2+
were loaded at 65.degree. C. (FIG. 4B) or 70.degree. C. (FIG. 4C).
Uptake was significantly faster at both temperatures, and maximum
loading was achieved after .about.20 min.
These data show that encapsulation in liposomes comprising EDHSM as
compared to SM is more efficient at a higher temperature, thereby
demonstrating that liposomes comprising EDHSM have decreased
membrane permeability than those composed of SM.
EXAMPLE 4
Characterization of In Vitro Drug Release from Liposomal
Compositions Comprising Various Sphingomyelins and
Dihydrosphingomyelin
The in vitro drug release rates of liposomal compositions
comprising vincristine encapsulated in liposomes comprising
sphingomyelin derived from different sources were determined and
compared. The various sources of sphingomyelin examined included
egg sphingomyelin, milk sphingomyelin, and brain sphingomyelin
obtained from Avanti Polar Lipids, Inc., as well as egg
dihydrosphingomyelin prepared as described in Example 1. All
liposomes comprised sphingomyelin and cholesterol at a 55/45 molar
ratio and a drug:lipid ratio of 0.1 (w/w).
Liposomes were loaded with vincristine using standard procedures,
including primarily the ionophore-mediated loading method as
described in Fenske, D. B. et al., Biochim Biophys Acta.
1414(1-2):188-204 (1998).
Drug release assays were performed at 50.degree. C. in 50% FBS, as
previously described in Fenske et al. 1998, except that fetal
bovine serum was used instead of mouse serum, and the temperature
was increased to 50.degree. C., from 37.degree. C. used in Fenske
et al. (1998). Drug retention was measured at various time points,
and the results of these studies are depicted in FIG. 5. These
results demonstrate that egg dihydrosphingomyelin dramatically
increases liposome drug retention and that liposomes comprising
DHSM possess remarkably superior properties for drug delivery as
compared to liposomes prepared using SM.
EXAMPLE 5
Characterization of In Vitro Drug Release from Liposomal
Compositions Comprising Various Dihydrosphingomyelin Species
The rate of vinorelbine release from liposomes comprising various
dihydrosphingomyelin species was compared in in vitro studies. Drug
release rates were also determined for liposomes comprising a
mixture of phospholipids, including egg dihydrosphingomyelin.
The lipids were dissolved in ethanol at 65.degree. C. to achieve a
final lipid concentration of 100 mg/ml. The hot lipid mixture was
then added as a steady stream by injection with a syringe to a 353
mM MgSO.sub.4/235 mM sucrose solution with mixing to form
multilamellar vesicles (MLV) at final lipid and ethanol
concentrations of 15 mg/ml and 15% (vol/vol), respectively. The MLV
were extruded at 65.degree. C. through two stacked 80 nm
polycarbonate membranes by applying nitrogen gas pressure
(.about.200 psi) to a 100 ml Extruder. Extrusion was repeated until
a vesicle size of 90 to 110 nm was achieved, which usually required
4 to 6 passes. However, only two passes were required for EPC/CH
vesicles. Vesicle size was determined by quasi-elastic light
scattering using a Nicomp 380 submicron particle sizer (Santa
Barbara, Calif.). The resulting large unilamellar vesicles were
dialyzed against 300 mM sucrose to remove residual ethanol and
external MgSO.sub.4 using a tangential flow filtration system (20
wash volumes). The final preparation was concentrated to .about.50
mg/ml, and an aliquot was analyzed for SM and EPC content by
phosphate assay. The vesicles were stored at 5.degree. C. until
required for loading.
Vinorelbine was loaded into the SM/CH (55:45 mole ratio) liposomes
using the A23187-ionophore method. EDTA and phosphate buffer were
added to the liposomes (15 mg/ml total lipid, pH 6.5) at final
concentrations of 25 mM and 50 mM, respectively. The liposome
suspensions were pre-heated to 60.degree. C. using a water bath
before addition of the ionophore (1 .mu.g A23187/mg lipid). After a
10 min incubation, a 10 mg/ml vinorelbine stock solution
solubilized 300 mM sucrose was added at a drug to lipid ratio of
0.417 (mol/mol). This molar drug to lipid ratio is equivalent to a
wt/wt ratio of 0.3 for ESM/CH vesicles. The solution was incubated
for 60 min at 60.degree. C. to induce vinorelbine encapsulation
after which the ionophore, EDTA and unencapsulated topotecan were
removed by tangential flow filtration using 20 wash volumes of 300
mM sucrose, 20 mM sodium phosphate, pH 6.5 buffer.
The same procedure was used for loading the EPC/CH and EPC/EDHSM/CH
IVR preparations but with the following changes. An incubation
temperature of 50.degree. C. was used instead of 60.degree. C. and
the ionophore A23187 was not pre-incubated with the vesicles but
added to the pre-warmed vesicles just prior to adding the
vinorelbine solution. This was to minimize loss of the pH gradient
due to an ionophore mediated Na.sup.+/H.sup.+ exchange.
Furthermore, a 10 and 40 min incubation time was used for the
EPC/CH and EPC/EDHSM/CH formulations, respectively.
Lipid (CH and ESM) and vinorelbine content was analyzed by HPLC.
For vesicles containing lipids other than CH or ESM, the total
lipid content was calculated based on a theoretical molar ratio of
45% for CH.
Vinorelbine release rates were compared using an in vitro release
(IVR) assay. The IVR assay was conducted using a release buffer of
7 mM NH.sub.4Cl, 10 mM Na.sub.2HPO.sub.4, 153 mM NaCl, pH 6.0.
Briefly, the liposomal vinorelbine formulations (.about.0.5 ml)
were diluted into 100 ml of release buffer to a final lipid content
of 0.36 .mu.mol, which is equivalent to 0.2 mg of ESM/CH (55:45 mol
ratio). The mixtures were incubated in a 37.degree. C. or
25.degree. C. water bath and at various times aliquots were
withdrawn and unencapsulated vinorelbine separated from the
liposomes using Microcon YM-100 (100 kDa MW cutoff) centrifugation
devices. Total and free vinorelbine content was measured by high
performance liquid chromatography (HPLC).
In FIG. 6 are shown vinorelbine release rates from liposomes
composed of ESM:Chol, EDHSM:Chol, BDHSM:Chol and MDHSM:Chol on
incubation in the IVR release buffer at 37.degree. C. Drug release
is slowest from liposomes composed of EDHSM and BDHSM, while
liposomes composed of MDHSM show a similar rate of drug release to
ESM liposomes. Without wishing to be bound by any particular
theory, it is noted that while EDHSM and BDHSM exhibit a narrow,
single phase transition by DSC (FIG. 1 and FIG. 3), MDHSM exhibits
a more complex thermotropic behavior (see FIG. 2). This may be
related to the fact that the N-acyl and dihydrosphingosine chain
lengths are similar in EDHSM and BDHSM (Table 1) while the majority
of N-acyl chains in MDHSM are longer than the dihydrosphingosine
chain by four carbon atoms or greater (Table 1). Again without
wishing to be bound by any particular theory, the slower
vinorelbine release rates seen for liposomes composed of EDHSM and
BDHSM may result from the similar dihydrosphingosine and N-acyl
chains lengths in these species.
In FIG. 7 are shown vinorelbine release rates from liposomes
composed of EPC:Chol and EPC:EDHSM:Chol on incubation in IVR
release buffer at 25.degree. C. As can be seen, the inclusion of
DHSM with EPC in liposomes results in slower vinorelbine release
compared to similar vesicles containing only EPC.
EXAMPLE 6
Comparison of In Vivo Drug Release from Liposomal Compositions
Comprising Egg Sphingomyelin or Egg Dihydrosphingomyelin
The in vivo drug release rates of compositions comprising various
drugs encapsulated in liposomes comprising either egg sphingomyelin
(ESM) or egg dihydrosphingomyelin (EDHSM) were determined and
compared. Liposomal compositions comprising either ESM or EDHSM
were injected into ICR mice as described in Webb, M. S. et al., Br
J Cancer 72(4):896-904 (1995) and Boman, N. L. et al., Cancer Res.
54(11):2830-3 (1994), and plasma drug retention was determined by
standard procedures at various time points following injection.
Drug retention rates for liposomal compositions comprising
vincristine, NK611, or topotecan are depicted in FIGS. 8A, 8B, and
8C, respectively.
Various drug:lipid ratios and dosages were examined, as indicated
in FIG. 8. Vincristine was administered at a dosage of 2 mg/kg;
drug:lipid ratios are indicated on the figure. NK611 was
administered at a dose of 20 mg/kg for both formulations (ESM/Chol,
D/L ratio 0.36; egg-DHSM/Chol, D/L ratio 0.34). Topotecan was
administered at a dose of 5 mg/kg for both formulations. In
addition, for the liposomal NK611 composition the liposomes were
prepared in 300 mM Mn.sup.2+, while the liposomal topotecan
compositions were prepared in magnesium at a concentration of 300
mM Mn.sup.2+. Drug loading was by the ionophore method using
A23187.
The results of these studies demonstrate that for each drug tested,
compositions comprising egg dihydrosphingomyelin (egg-DHSM)
exhibited markedly increased drug retention as compared to
compositions comprising egg sphingomyelin (ESM) and, therefore,
possess superior characteristics for in vivo drug delivery.
EXAMPLE 7
Pharmacokinetic Properties of Liposomal Topotecan Formulations
Comprising Dihydrosphingomyelin Loaded Using MG.sup.2+ or
MN.sup.2+
Pharmacokinetic (PK) studies were conducted to characterize
liposomal formulation of topotecan comprising SM or DHSM. In
addition, the pharmacokinetic performance of SM and DHSM
formulations were compared when topotecan was loaded using the
ionophore method with intravesicular Mn.sup.2+ or Mg.sup.2+.
Liposomal topotecan formulations were prepared and loaded
essentially using an ionophore method as originally described in
Fenske et al., Biochim Biophys Acta 1414(1-2): 188-204 (1998).
Lipids (ESM or DHSM and cholesterol) were dissolved in ethanol at
65.degree. C. to achieve a final lipid concentration of 178 mM
(equivalent to 100 mg/ml of the standard ESM/CH (55:45 mol ratio)
formulation). For formulations used in animal studies, trace
amounts the radiolabeled lipid .sup.3H-cholesteryl hexadecyl ether
(.sup.3H-CHE, 6 .mu.mol/mol lipid; 0.55 .mu.Ci/mg lipid) were
included in the lipid mixture. The .sup.3H-CHE was added by first
drying off the toluene solvent under a stream of nitrogen gas then
re-suspending the .sup.3H-CHE in the ethanol-lipid solution.
Multilamellar vesicles (MLV) were formed by adding the hot lipid
solution as a steady stream by injection with a syringe over
.about.30 seconds with mixing to a 353 mM MgSO.sub.4/235 mM sucrose
or 353 mM MnSO.sub.4/235 mM sucrose solution.
The MLV were extruded at 65.degree. C. through two stacked 80 nm
polycarbonate membranes by applying nitrogen gas pressure
(.about.200 psi) to a 10 or 100 ml Extruder. Extrusion was repeated
until a vesicle size of 90 to 110 nm was achieved, which usually
required 4 to 6 passes. Vesicle size was determined by
quasi-elastic light scattering using a Nicomp 380 submicron
particle sizer (Santa Barbara, Calif.).
The resulting large unilamellar vesicle (LUV) formulation was
dialyzed against 300 mM sucrose to remove the residual ethanol and
external magnesium sulphate using a tangential flow ultrafiltration
system (20 wash volumes). The final preparation was concentrated to
50 mg/ml and stored at 5.degree. C. until required for loading. The
lipid concentration of the radiolabeled formulations for the animal
study were determined from the SM content measured by using a
phosphate assay. The total lipid concentration was calculated from
this value by using the target mole percent of SM to CH
(55:45).
Topotecan was loaded into liposomes using the A23187-ionophore
method. EDTA and phosphate buffer were added to liposomes (15 mg/ml
total lipid, pH 6.5) at 25 mM and 50 mM final concentrations,
respectively. The liposome suspensions were pre-heated to
60.degree. C. using a water bath before the ionophore (0.5 .mu.g
A23187/mg lipid) was added. After a 10 min incubation, a 10 mg/ml
topotecan stock solution (solubilized in 1 mg/ml tartaric acid) was
added. The solution was typically incubated for 60 min at
65.degree. C. to induce topotecan encapsulation after which the
ionophore, EDTA and unencapsulated topotecan were removed by
tangential flow diafiltration against phosphate buffered sucrose
(300 mM sucrose, 10 mM sodium phosphate, pH 6). Samples were then
filtered through a 0.45 .mu.m pore-size syringe filter followed by
passage through a 0.22 .mu.m filter before use.
All vesicles contained .sup.3H-CHE to allow monitoring of the lipid
clearance rates and calculation of drug-to-lipid ratios to measure
drug payout in vivo.
The loading efficiency, final drug-to-lipid (D/L) ratio and vesicle
size of the formulations are shown in Table 3.
TABLE-US-00003 TABLE 3 Summary of Liposomal Topotecan Formulations
Lipid Internal cation Vesicle Loading composition used for size
Final D/L efficiency (mol ratio) loading (dia., nm) ratio (wt/wt)
(%) ESM/CH (55:45) Mg.sup.2+ 100 .+-. 24 0.109 94 DHSM/CH Mg.sup.2+
110 .+-. 20 0.109 92 (55:45) ESM/CH (55:45) Mn.sup.2+ 110 .+-. 10
0.103 100 DHSM/CH Mn.sup.2+ 100 .+-. 20 0.104 98 (55:45)
Mice were dosed at 50 mg lipid/kg via the lateral tail vein. Volume
of injection was based on the body weight of the individual mouse
(200 .mu.l/20 g mouse). For each formulation, 4 mice were dosed per
time point. Animals were anesthetized with ketamine/xylazine at
0.5, 2, 4, 8, and 16 hours, and blood was harvested by cardiac
puncture. Blood was collected into EDTA vacutainer tubes and 50
.mu.l aliquots of whole blood sample were removed for topotecan and
lipid analysis. Plasma was separated by centrifugation (250.times.g
for 10 minutes) and 50 .mu.l aliquots were analyzed by fluorescence
assay for topotecan and liquid scintillation counting for lipid,
respectively.
Topotecan was recovered from blood and plasma by extracting 50
.mu.l blood or plasma with 600 .mu.l of cold methanol. Samples were
then centrifuged at 13,400.times.g for 3 minutes, and 100 .mu.l of
the supernatant was removed and diluted in 700 .mu.l methanol and
200 .mu.l TRIS (50 mM, pH 8). Samples were measured against a
standard curve (0 to 500 ng) in which blood or plasma (50 .mu.l)
was spiked with topotecan and extracted using the same protocol.
Fluorescence was measured using a SLM Aminco Bowman Series 2
Luminescence Spectrometer at an excitation wavelength of 380 nm
with a 2 nm band pass and an emission of 518 nm with a band pass of
4 nm.
Lipid recovery was measured with radiolabeled .sup.3H-CHE. A 50
.mu.l aliquot of blood or plasma sample from each condition was
transferred to a glass scintillation vial for digestion and
decolorization. For the digestion, 500 .mu.l of Solvable was added
to each vial and kept overnight at ambient temperature in the dark
(>16 h). Then to decolorize, the following reagents were added:
50 .mu.l of 200 mM EDTA (pH 7.5), 200 .mu.l of 30% hydrogen
peroxide (added cold from the fridge), and 25 .mu.l of 10 N HCl.
Sample vials were capped loosely and stored at room temperature in
the dark overnight (16 h). Next, 5 ml of Pico Flour 40 was added to
all vials, which were capped tightly and inverted in order to
thoroughly mix the samples. All samples were loaded onto the
scintillation counter (Beckman LS 6500) and measured.
Pharmacokinetic profiles for these liposomal topotecan formulations
are shown as a percentage of the starting values for the injected
material at different time points (FIG. 9). Key pharmacokinetic
parameters (AUC and T.sub.1/2) were calculated from these plots and
the data are presented in Table 4.
TABLE-US-00004 TABLE 4 Key Pharmacokinetic Parameters for Various
Liposomal Topotecan Formulations T1/2 Upper-lower 95% conf. Limit
AUC .+-. S.E..sup.1 (h) (h) BLOOD Drug Retention AUC (h * %) ESM/CH
- Mg 484 .+-. 16 2.6 2.0-3.8 DHSM/CH - Mg 842 .+-. 16 5.3 4.0-7.7
ESM/CH - Mn 879 .+-. 34 5.0 3.4-9.6 DHSM/CH - Mn 1093 .+-. 19 9.0
5.4-27.1 TOPOTECAN AUC (h * .mu.g/mL) ESM/CH - Mg 199 .+-. 5 2.1
1.8-2.6 DHSM/CH - Mg 289 .+-. 7 3.6 3.2-4.2 ESM/CH - Mn 299 .+-. 15
3.3 2.4-5.1 DHSM/CH - Mn 375 .+-. 12 5.5 4.0-8.4 LIPID AUC (h *
mg/mL) ESM/CH - Mg 4.9 .+-. 30 12.4 8.2-25.2 DHSM/CH - Mg 4.9 .+-.
20 11.9 7.8-24.9 ESM/CH - Mn 4.4 .+-. 0.1 9.6 8.2-11.6 DHSM/CH - Mn
5.2 .+-. 0.2 13.7 11.3-17.4 PLASMA Drug Retention AUC (h * %)
ESM/CH - Mg 456 .+-. 19 2.5 2.1-3.2 DHSM/CH - Mg 752 .+-. 26 5.0
4.1-6.6 ESM/CH - Mn 831 .+-. 40 4.3 3.3-6.0 DHSM/CH - Mn 999 .+-.
41 8.1 5.9-12.7 TOPOTECAN AUC (h * .mu.g/mL) ESM/CH - Mg 319 .+-. 9
2.1 1.7-2.8 DHSM/CH - Mg 461 .+-. 17 3.7 3.0-4.9 ESM/CH - Mn 517
.+-. 28 3.1 2.3-4.5 DHSM/CH - Mn 646 .+-. 26 5.2 3.8-8.0 LIPID AUC
(h * mg/mL) ESM/CH - Mg 9.3 .+-. 0.4 14.1 9.5-27.2 DHSM/CH - Mg 9.1
.+-. 0.1 14.4 9.9-26.6 ESM/CH - Mn 8.8 .+-. 0.3 11.4 8.5-17.3
DHSM/CH - Mn 10.2 .+-. 0.3 14.2 10.4-22.5 .sup.1Standard Error
Liposomes comprising egg SM (ESM) and cholesterol and loaded with
topotecan using a Mg.sup.2+ ion gradient exhibited the fastest
topotecan release rate (FIGS. 9A, 9D and Table 4;
T.sub.1/2.about.2.6 h). Similar liposomes loaded with topotecan
using Mg.sup.2+ but composed of egg dihydrosphingomyelin (DHSM),
show slower drug release (FIGS. 9A, 9D and Table 4) and exhibit a
corresponding increase in AUC for drug retention and a longer drug
release half-life (Table 4). Similarly when liposomes composed of
ESM and DHSM but loaded with topotecan using Mn.sup.2+ are
compared, drug release is also slower for the DHSM formulation
(FIGS. 9A and 9B). Again the DHSM liposomes exhibit a higher AUC
for drug retention and a longer drug release half-life (Table
4).
At the lipid doses used in this study, the rate of liposome
clearance from the blood/plasma compartment was not significantly
different for liposomes composed of ESM or DHSM (FIGS. 9B and 9E
and Table 4) over 16 hours. However, as shown in Example 9, at
lower lipid doses, liposomes composed of DHSM are cleared from the
blood compartment more slowly than similar liposomes composed of
ESM.
The reduced rate of topotecan release from liposomes composed of
DHSM is also reflected in the topotecan pharmacokinetics. Blood and
plasma drug levels are higher for the two DHSM formulations
(Mn.sup.2+ and Mg.sup.2+) compared to their corresponding ESM
counterparts (FIGS. 9C and 9F). This results in higher drug AUCs
and longer drug circulation half-lives for the DHSM formulations
compared to the ESM formulations (Table 4).
In summary, these data indicate that liposomal drug formulations
comprising DHSM possess surprisingly superior pharmacokinetic
properties in comparison to previously described liposomal drug
formulations.
EXAMPLE 8
Pharmacokinetic Properties of Liposomal Vinorelbine Formulations
Comprising Dihydrosphingomyelin Loaded Using MG.sup.2+ or
MN.sup.2+
Pharmacokinetic (PK) studies were conducted to characterize
liposomal formulations of vinorelbine comprising SM or DHSM. In
addition, the pharmacokinetic performance of SM and DHSM
formulations were compared when vinorelbine was loaded using the
ionophore method with intravesicular Mn.sup.2+ or Mg.sup.2+.
Liposomes of ESM or DHSM were prepared as described in Example 5
containing either Mn.sup.2+ or Mg.sup.2+ as the intravesicular
cation. Vinorelbine was loaded into the liposomes using the
ionophore method with A23187, as described in Example 5, at
drug/lipid ratios of either 0.1 or 0.3 (target ratios).
The pharmacokinetic properties of the liposomal vinorelbine
formulations were determined in ICR mice as described for liposomal
topotecan in Example 7. Liposome and vinorelbine concentrations in
blood and plasma were determined by liquid-scintillation counting
for .sup.3H-CHE (liposome concentration) and by HPLC analysis
(vinorelbine concentration).
Pharmacokinetic profiles for these liposomal vinorelbine
formulations were determined and key pharmacokinetic parameters
(AUC and T.sub.1/2) were calculated from these plots (Table 5).
TABLE-US-00005 TABLE 5 Key Pharmacokinetic Parameters (Plasma) for
Various Liposomal Vinorelbine Formulations T1/2 Initial Upper-lower
Drug:Lipid 95% conf. Limit Ratio AUC .+-. S.E..sup.1 (h) (h)
Drug:Lipid Ratio 0.31 Drug Retention AUC (h * %) ESM/CH - Mg 1130
.+-. 24 8.6 6.8-11.9 DHSM/CH - Mg 1564 .+-. 22 12.7 8.6-24.6 ESM/CH
- Mn 1429 .+-. 37 8.0 5.1-19.2 DHSM/CH - Mn 1948 .+-. 21 19.5
11.8-55.4 VINORELBINE AUC (h %) ESM/CH - Mg 584 .+-. 27 4.1 3.1-5.8
DHSM/CH - Mg 713 .+-. 26 5.3 4.6-6.2 ESM/CH - Mn 687 .+-. 22 4.3
3.2-6.7 DHSM/CH - Mn 860 .+-. 27 5.8 4.4-8.7 LIPID AUC (h * %)
ESM/CH - Mg 707 .+-. 30 7.6 5.6-12.0 DHSM/CH - Mg 656 .+-. 25 8.9
7.1-12.1 ESM/CH - Mn 692 .+-. 30 11.0 8.8-14.6 DHSM/CH - Mn 650
.+-. 20 8.2 6.0-12.9 Drug:Lipid Ratio 0.1 Drug Retention AUC (h *
%) ESM/CH - Mg 876 .+-. 18 6.6 5.0-9.6 DHSM/CH - Mg 1067 .+-. 11
11.0 7.2-23.2 ESM/CH - Mn 1266 .+-. 11 14.9 9.6-32.4 DHSM/CH - Mn
1522 .+-. 46 31.3 17.2-177.0 VINORELBINE AUC (h * .mu.g/mL) ESM/CH
- Mg 1842 .+-. 50 3.9 2.9-6.0 DHSM/CH - Mg 1856 .+-. 31 6.2 5.2-7.8
ESM/CH - Mn 2477 .+-. 80 6.5 5.3-8.6 DHSM/CH - Mn 2481 .+-. 71 8.3
5.8-14.6 LIPID AUC (h * mg/mL) ESM/CH - Mg 9.02 .+-. 0.22 10.1
7.4-15.7 DHSM/CH - Mg 7.92 .+-. 0.18 14.4 11.7-18.6 ESM/CH - Mn
8.50 .+-. 0.32 11.3 9.5-13.9 DHSM/CH - Mn 7.68 .+-. 0.18 10.9
8.2-16.2 .sup.1Standard Error
Liposomal formulations of vinorelbine comprising DHSM showed slower
drug release in vivo, as indicated by higher drug retention AUC
values and longer drug release half-lives, compared to similar
liposomes comprised of ESM (Table 5). This improved vinorelbine
retention by DHSM liposomes compared to ESM liposomes was seen for
formulations loaded at a 0.1 drug:lipid ratio and formulations
loaded at a 0.31 drug:lipid ratio. Similarly, DHSM liposomes loaded
with vinorelbine using either Mn.sup.2+ or Mg.sup.2+, showed slower
vinorelbine release in vivo compared to similar ESM liposomes
loaded using either Mn.sup.2+ or Mg.sup.2+ (Table 5).
In summary, these results surprisingly demonstrate that DHSM
liposomes provide slower release of encapsulated vinorelbine
compared to ESM liposomes over a wide range of drug:lipid ratios
and using different loading procedures.
EXAMPLE 9
Plasma Circulation Half-Life of Dihydrosphingomyelin Liposomes
Liposomes comprising ESM:cholesterol or DHSM:cholesterol were
prepared using similar procedures to those described in Examples 7
and 8, and incorporating the radiolabeled lipid marker .sup.3H-CHE.
The plasma residency of these liposomes after intravenous injection
into mice was then compared at two lipid doses (25 mg/m.sup.2 and
250 mg/m.sup.2).
As shown in FIG. 10, DHSM liposomes remain in the plasma
compartment longer than similar liposomes containing ESM,
particularly at lower lipid doses and at longer timepoints. For
drug delivery applications, circulation lifetime is important
because the longer the liposomes remain in the plasma compartment,
the greater the likelihood that they will accumulate at a disease
site, for example within a tumor. A longer circulation lifetime
can, therefore, result in greater drug delivery to the disease
site.
These results demonstrate that in addition to providing slower drug
release in vivo, DHSM liposomes also exhibit a longer circulation
half-life compared to similar liposomes composed of ESM. This
unexpected property can further enhance the pharmacokinetic
behaviour of drugs encapsulated in liposomes of the present
invention.
EXAMPLE 10
Antitumor Activity of Liposomal Topotecan Formulations Comprising
Dihydrosphingomyelin
The antitumor activity of liposomal formulations of topotecan
comprising ESM or DHSM were evaluated in human tumor xenograft
models having significant (MX-1 (breast)) and modest (HT-29
(colon)) sensitivity to free topotecan at its maximum therapeutic
dose (MTD). These studies also evaluated the influence of
intravesicular cation composition (Mn.sup.2+ or Mg.sup.2+) on
antitumor activity.
Liposomal formulations comprising ESM or DHSM and containing
topotecan were prepared as described in Example 7. Topotecan
loading was conducted with either Mg.sup.2+ or Mn.sup.2+ as the
intravesicular cation.
DHSM/Chol (55/45, mol/mol) and ESM/Chol (55/45, mol/mol) liposome
formulations were prepared and loaded using an ionophore method as
originally described in Fenske et al., Biochim Biophys Acta
1414(1-2): 188-204 (1998) and described in more specific detail in
Example 4. Liposomes (15 mg/mL) containing 200 mM sucrose and
.about.300 mM MnSO.sub.4 or MgSO.sub.4 were incubated with A23187
ionophore (0.5 .mu.g/mg of lipid) in 300 mM sucrose, 25 mM EDTA,
and 50 mM phosphate buffer (pH 6.0). This mixture was warmed for 10
min at 65.degree. C. Topotecan (10 mg/ml in 1 mg/mL tartaric acid,
300 mM sucrose) was added to achieve a drug-to-lipid ratio of 0.1
(wt/wt) and drug loading occurred during a 60 min incubation at
65.degree. C. To remove non-encapsulated topotecan, ionophore and
EDTA, the incubation mixture was dialyzed at room temperature
against 20 volumes of phosphate-buffered sucrose (10 mM sodium
phosphate, 300 mM sucrose; pH 6.0) using a Midgee.TM. HOOP.TM.
ultrafiltration cartridge (M.Wt. cutoff 100,000; Amersham
Biosciences). Samples were filtered through a 0.22 .mu.m filter
prior to vialing.
Liposomal topotecan formulations were diluted to the appropriate
drug concentration using sterile 10 mM sodium phosphate, 300 mM
sucrose, pH 7.4, in preparation for animal injections. All
formulations were injected intravenously via the lateral tail vein
of female, 6-8 week old, athymic Crl:CD-1.RTM.-nuBR mice obtained
from Charles River Laboratories (Quebec, Canada). Experimental
groups consisted of 8 or 5 mice for MX-1 and HT-29 studies,
respectively. The dosing volume of each formulation was 10 mL/kg
body weight. For the study with MX-1 xenografts, sample injections
were q7d.times.3, beginning on day 13 post-tumor implantation. For
the HT-29 xenograft study, sample injections were q4d.times.3,
beginning on day 9 post-tumor implantation.
MX-1 human mammary carcinoma tumor fragments were obtained from the
Division of Cancer Treatment and Diagnosis (DCTD) Tumor Repository
(Frederick, Md.), maintained by serial passage in vivo, and
implanted by trocar into the dorsal flank of the nude mice. HT-29
human colon adenocarcinoma cells were obtained from the American
Type Culture Collection (ATCC; Manassas, Va.) and maintained in
vitro in McCoy's 5A medium supplemented with 10% fetal bovine serum
and 2 mM L-glutamine. On day 0, tumor cells (5.times.10.sup.6) were
implanted via subcutaneous (s.c.) injection into the dorsal flank.
Treatments were initiated when tumor volumes were 100-300 mm.sup.3.
Tumors were measured at least three times per week with calipers
and tumor volume (mm.sup.3) was calculated using the formula:
(length.times.width.sup.2)/2, where width was the smaller of the
two perpendicular measurements (Fiebig H-H and Burger A M, In:
Tumor Models in Cancer Research (Ed. Teicher B A), pp. 113-137.
Humana Press Inc, Totowa (2002).
Therapeutic activity was evaluated by several criteria, as
discussed in detail in Plowman et al. In: Anticancer Drug
Development Guide: Preclinical Screening, Clinical Trials, and
Approval (Ed. Teicher B). Humana Press Inc., Totowa (1997).
Calculated tumor parameters included: (i) tumor growth delay (T-C);
the mean difference in time (days) for treated and control tumors
to reach 1000 mm.sup.3; (ii) partial regressions (PR) and complete
regressions (CR); a PR was scored when a tumor decreased to
.ltoreq.50% of its initial size but remained above the limit of
measurability (63 mm.sup.3); a CR was scored when a tumor regressed
below 63 mm.sup.3 but ultimately showed regrowth; and (iii) tumor
free animals (TF) were scored when a tumor regressed below 63
mm.sup.3 and remained below this level up to and including the
final observation day.
In FIG. 11 are shown tumor growth rates in the MX-1 model for
untreated control animals and for animals treated with the
liposomal topotecan formulations. Antitumor activity parameters are
summarized in Table 6.
TABLE-US-00006 TABLE 6 Summary of antitumor activity parameters -
MX-1 mammary xenografts Topotecan Topotecan Dose Internal T-C
PR/CR/TF Formulation (mg/kg) Cation (days) n = 8 ESM/Chol 1.0
Mg.sup.2+ 7.6 0/0/0 ESM/Chol 0.5 Mg.sup.2+ 8.2 0/0/0 DHSM/Chol 1.0
Mg.sup.2+ 33.8 1/2/1 DHSM/Chol 0.5 Mg.sup.2+ 5.8 0/0/0 ESM/Chol 1.0
Mn.sup.2+ 14.3 0/1/0 ESM/Chol 0.5 Mn.sup.2+ 10.4 0/0/0 DHSM/Chol
1.0 Mn.sup.2+ 57.7 0/4/4 DHSM/Chol 0.5 Mn.sup.2+ 24.9 1/2/0
Antitumor activities are consistent with the pharmacokinetic
properties of these formulations. When topotecan is loaded using
Mg.sup.2+ a significantly longer delay in tumor growth (T-C) is
seen for the DHSM formulation at both 1.0 and 0.5 mg/kg compared to
the ESM formulation at these same doses (Table 6). Further, only in
the DHSM formulation (1 mg/kg) is a tumor-free animal seen, as well
as complete and partial tumor responses. Similarly, for
formulations loaded using Mn.sup.2+ as the intravesicular cation,
tumor growth delays were considerably longer for the DHSM
formulation compared to the ESM formulation. Further, only for the
DHSM formulation (1 mg/kg) were all treated animals either complete
responders (CR) or tumor free survivors (TF) (Table 6). This study
also evaluated the toxicities of these liposomal topotecan
formulations based on animal weight loss. As shown in FIG. 12, no
significant weight loss was seen in any treatment group. This
indicates that, in addition to showing good antitumor activity, the
DHSM formulations were well tolerated.
Antitumor activity was also evaluated in the HT-29 colon xenograft
model. In this study, animals were treated either with liposomes
comprising ESM loaded with topotecan using Mg.sup.2+ or liposomes
comprising DHSM loaded with topotecan using Mn.sup.2+. Animals
treated with the ESM/Chol/Mg.sup.2+ formulation of liposomal
topotecan in this model showed marginal or modest activity after
i.v. injection using a q4d.times.3 dosing schedule (FIG. 13). At
the highest dose (4.0 mg/kg/dose), T-C was 29.3 days and two
partial and three complete responses were observed out of five mice
(Table 7). The DHSM/Chol/Mn.sup.2+ formulation showed improved
antitumor activity in this model, with a T-C of 37.2 days at 4.0
mg/kg (q4d.times.3, i.v.) and four complete responses and one tumor
free animal out of five mice. Partial and complete responses were
also observed in the next two dosing groups (1.0 and 2.0
mg/kg/dose).
TABLE-US-00007 TABLE 7 Summary of antitumor activity parameters -
HT-29 colon xenograft model Topotecan Topotecan Dose Internal T-C
PR/CR/TF Formulation (mg/kg) Cation (days) n = 5 ESM/Chol 4.0
Mg.sup.2+ 29.3 2/3/0 ESM/Chol 2.0 Mg.sup.2+ 21.3 0/0/0 ESM/Chol 1.0
Mg.sup.2+ 13.4 0/0/0 ESM/Chol 0.5 Mg.sup.2+ 4.8 0/0/0 DHSM/Chol 4.0
Mn.sup.2+ 37.2 0/4/1 DHSM/Chol 2.0 Mn.sup.2+ 27.3 2/3/0 DHSM/Chol
1.0 Mn.sup.2+ 22.6 0/2/0 DHSM/Chol 0.5 Mn.sup.2+ 63.1 0/0/0
Treatment-related changes in total body weight were monitored
during the dosing phase. Static or increasing mean group weights
were observed at the two lowest treatment doses for both liposomal
topotecan formulations examined. In the HT-29 model, which was
dosed more aggressively at q4d.times.3, a significant and
progressive decrease in weight (.about.23%) was observed during the
dosing phase with the high dose (4.0 mg/kg/dose)
DHSM/Chol/Mn.sup.2+ formulation (FIG. 14). In contrast, the same
dose and schedule for the ESM/Chol/Mg.sup.2+ formulation resulted
in a maximum group weight loss of .about.6%. The increased weight
loss for the DHSM/Chol/Mn.sup.2+ formulation is consistent with
increased drug retention for this formulation and maintenance of
the drug in the active lactone form, compared with the
ESM/Chol/Mg.sup.2+ formulation. All other dosing levels and
formulations examined in this study exhibited maximum weight losses
<5% or showed progressive weight gain.
In summary, the improved antitumor activity of liposomal topotecan
comprising DHSM in both MX-1 and HT-29 xenografts demonstrates that
a higher plasma topotecan AUC results in an improved efficacy
profile against human xenograft models and supports the clinical
efficacy of liposomal topotecan formulations comprising DHSM.
All of the above U.S. patents, U.S. patent application
publications, U.S. patent applications, foreign patents, foreign
patent applications and non-patent publications referred to in this
specification and/or listed in the Application Data Sheet, are
incorporated herein by reference, in their entirety.
From the foregoing it will be appreciated that, although specific
embodiments of the invention have been described herein for
purposes of illustration, various modifications may be made without
deviating from the spirit and scope of the invention. Accordingly,
the invention is not limited except as by the appended claims.
* * * * *
References